Essay: Proteins

INTRODUCTION
Proteins are one the most common and important cellular macromolecules, which controls almost all biological processes. The functionality of a polypeptide chain depends on the attainment of a three- dimensional compact structure after its synthesis at the ribosome (Dobson 2003 nature 426: 884-890). Globular proteins can interact with their molecular targets only when it attains perfectly folded conformation (2). The stability of a protein in the complex biological environment also depends upon its correct folding and conformation. Any small error during folding process of a protein may lead to misfolding and formation of aggregates leading to cellular stress and cell death. Therefore, misfolding leads to many neurodegenerative diseases like cystic fibrosis, Alzheimer’s disease, Creutzfeldt- Jakob disease, Parkinson’s disease and familial amyloidotic polyneuropathy [Dobson, 2002; Hammarstrom et al., 2002; Thomas et al., 1995]
Protein folding in vitro and in vivo, takes place in a different manner. There are a number of proteins inside the cell like peptidyl prolyl isomerase (PPI), protein disulfide isomerase (PDI) and many chaperones [Bukau et al, 1996] that assists in folding process and prevents misfolding of the proteins. However, to understand the role of such auxiliary proteins, knowledge of unassisted folding is required, therefore the focus on in vitro experiments.
Although bacteria itself has some defense mechanisms by which it can protect native proteins from aggregation or misfolding caused by osmotic stress, which include the intracellular accumulation by uptake of osmolytes (Yancey et al., 2001). Cells have two important and effective mechanisms to survive under stress and to reduce aggregation by using molecular chaperones and chemical chaperone also known as osmolytes (Saunders et al., 2000). Osmolytes accumulated in the cells enhance protein folding and stability of the native state of proteins. It comprises of small molecule of organic compounds which are water-soluble include electrolytes and non-electrolytes and are everywhere in the living system.
The transition from unfolded to folded state of a protein may results in the formation of partially folded intermediate states that have a strong tendency to aggregate. Studying the partially folded intermediates formed during the process of protein folding is prerequisite for understanding the complex process of protein folding. It is also important to study the development of structural changes occurring during refolding of the protein (Gruebele, 2002; Pradeep and Udgaonkar, 2004; Sridevi et al., 2004). Many efforts have been made to characterize folding intermediates by analysing conformational changes occurring when protein pass from unfolded state to folded state. Unfolding of small globular proteins usually explained by a two-state transition, which does not involve intermediate species during unfolding process[10],[11]. However, there are many studies reporting the existence of intermediates during unfolding/ refolding of small proteins as well[12],[13],[14]. It has also been observed that, environmental condition plays a critical role in unfolding behaviour of a protein molecule like under different set of conditions various states may exists that may be different from native or completely unfolded state. Thus, it is necessary to detect and characterize intermediates in order to understand how a protein folds correctly and how it avoids misfolding. For thermodynamic measurements, it is necessary that unfolding reaction should attain equilibrium as well as shows reversibility in a path independent manner. Therefore, analyzing an equilibrium unfolding process of a protein and to monitor its reversibility by refolding study are mandatory to develop understanding regarding conformational properties of a protein.
Such study may also involve the formation of inclusion bodies due to very high concentration of protein in the cell during over-expression and protein purification. Because of these inclusion bodies, many enzymes or proteins of therapeutic and industrial application has to compromise with its functionality and stability. It is well known fact that proteins are sensitive to their environment as they are prone to aggregation, so if we can somehow increase the stability or shelf-life of these pharmaceutically important proteins or enzymes, it would definitely enhance the technological applications of such product [Wang, 1999]. Thus, to develop highly efficient and cost effective methods to enhance the stability of these important biomolecules against various environmental perturbations is the most challenging task. In order to accomplish this, the inactive protein in the inclusion bodies must be converted into biologically active conformation by in vitro renaturation. This may be favoured by using perfectly optimized environmental conditions. Optimization of in vitro renaturation conditions is highly crucial for efficient refolding of the protein. Refolding buffers containing redox agents along with manipulation of time is an important factor for the correct refolding of proteins containing free cysteine and disulphide bonds. For the purpose, most commonly used redox agents are oxidized and reduced glutathione[8]. Refolding of proteins to correct 3D conformation is a complex process and is poorly understood. By controlling the renaturation process through prevention of aggregation may favours the correct refolding of protein, leading to higher yield of the functional form of protein. The process of refolding of sulfhydryl containing proteins can be optimized by use of appropriate redox systems in the renaturation buffers along with stabilizers like glycerol[9].
Here, in the present study, we have choosen Dihydrofolate reductase (DHFR) enzyme as our model protein system. DHFR (EC 1.5.1.3.), is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as an electron donor. DHFR is an important pharmacological target and is a very good model for the study of enzyme structure/ function relationships because of its small size, availability of purified enzyme and well developed functional assay[20]. Here, biophysical characterization of an unexplored variant of DHFR i.e. Zebrafish DHFR (zDHFR) has been reported. zDHFR can be used in research as an alternative to mammalian species. It has high sequence similarity with humans[21]. There is no report of equilibrium unfolding of zDHFR available in the literature so far. Thus equilibrium unfolding study and investigation of refolding process of zDHFR would provide added information on DHFR folding and comparative information about folding unfolding behavior of various species of DHFR proteins.
A protein, differing in origin, may exhibit variable physicochemical behavior, difference in sequence homology, fold and function. Thus, studying structure-function correlationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence-structure-function correlationship. So, it has also been attempted to compare the physicochemical behavior of E. coli and human DHFR in relation to equilibrium unfolding in presence of various osmolytes and finally the conformational parameters of all the three variants of DHFR were compared.
Based on the above approach, the objectives of the present study were designed as follows:
1. Over-expression of various recombinant proteins in Escherichia coli.
2. Understanding the status of their cellular folding.
3. Purification of selected recombinant proteins.
4. Functional studies of recombinant proteins.
5. Conformational studies of recombinant proteins using Biochemical and Biophysical methods.
6. Deducing structure-function co-relationship of recombinant proteins.
CHAPTER 2
REVIEW OF LITERATURE
2.1. Protein Structure and Its Physical Basis
The functionality of a protein can only be governed by its three dimensional structure. The unique conformation acquired by a protein due to its shape, charge and hydrobhobicity gives it a particular surface pattern which is responsible for its specific and diversified functions. A unique 3D structure of the nacent polypeptide chain is responsible for the formation of this surface pattern. The amino acid sequence of a given protein determines the native state as well as the folding pathway of that protein. (Anfinsen 1973, ellis et al 1998).
Protein structure has following four levels:
Primary Structure: Primary structure comprises of a linear chain of amino acids which are covalently bonded. Some non- covalent interactions are also present in higher order structures of protein.
Secondary Structure: Polypeptides can fold into a number of regular structures. Globular proteins have secondary structures comprises of right-handed alpha helix (‘) and beta sheet (��). The right-handed ‘- helix has 3.6 amino acids per turn and is stabilized by hydrogen bonds between peptide N-H and C=O groups three residues apart. Parallel and antiparallel ��- sheets are stabilized by hydrogen bonds between different portions of the polypeptide chain. Other irregular secondary structures of the protein are known as random coil. These structures are responsible for structural flexibility of the proteins. X-ray crystallography, NMR and circular dichorism (CD) spectroscopy methods are frequently used for determining secondary structure of a protein.
Tertiary Structure: The different sections of secondary structure and connecting regions fold into a well- defined tertiary structure, with hydrophilic amino acids mostly on the surface and hydrophobic ones in the interior. This hydrophobic effect is the driving force of the protein folding process. The structure is stabilized by noncovalent interactions and sometimes disulphide bonds between cysteines. Such arrangement allows protein to fold into their biologically active native structures (3). Conformation provided by the tertiary structure brings various functional groups in close proximity which establishes binding sites and helps in various interactions.
Quaternary Structure: When two or more polypeptide chains come together to form a single functional protein, this association is known as quaternary structure. Not all proteins have quaternary structure. They are stabilized by non-covalent interactions like hydrogen bonding, van der Waals and ionic interactions between the subunits Intermolecular disulfide bonds are also responsible for stabilizing quaternary structures of
proteins.
2.2. Physical Forces involved in Protein Folding and Structure
A linear polypeptide chain is autonomously organized into a space’filling, compact, and well’defined three dimensional structure. In a globular protein, the internal core is mostly formed by hydrophobic amino acid residues, held together by van der Waals forces, and the surface of the globule is formed by mostly charged and polar side chains. Proteins exist in this state of condensed matter, while the specific conformation is largely determined by the flexibility of the polypeptide backbone and by the specific, consistent intermolecular interactions of the side chains. The monomeric unit in a polypeptide chain is the peptide group. The sequence of amino acids is the primary structure of the protein. The C, O, N, and H atoms lie in the same plane; successive planes define angles f and c. The conformation of a chain of n amino acids can be defined by 2n parameters. The restricted flexibility of the polypeptide chain is a major factor among those determining protein structure and folding. The native conformation must be energetically stable. From a thermodynamic point of view, the free energy of a protein molecule is influenced by the following major contributions:
(1) the hydrophobic effect,
(2) the energy of hydrogen bonds,
(3) the energy of electrostatic interactions, and
(4) the conformational entropy due to the restricted motion of the main chain and the side chains.
The hydrophobic effect used to be explained as a primarily entropic effect (entropy is a measure of the number of specific ways in which a thermodynamic system may be arranged, commonly understood as a measure of disorder) arising from the rearrangement of hydrogen bonds between solvent molecules around an apolar solute. This hydration process is energetically unfavorable, and therefore drives apolar solutes together, thereby decreasing their solvent exposed surface area. Today, the hydrophobic effect is usually viewed as a combined effect of hydration (an entropic effect) and van derWaals interactions between solute molecules (an enthalpic effect) (Makhatadze and Privalov, 1995). It is therefore entropic at low temperatures and enthalpic at high temperatures, which results in a complex temperature dependence of its strength (Schellman, 1997). Nevertheless, the hydrophobic force has long been considered as the major driving force of protein folding (Dill, 1990) as it leads to a rapid collapse of the polypeptide chain, thereby largely reducing the configurational space to explore. Without doubt, the hydrophobic interaction is also a major stabilizing force contributing to the thermodynamic stability of the folded state. The role of hydrogen bonds in folding and stability used to be underestimated based on the argument that intramolecular hydrogen bonds can be replaced by hydrogen bonds between the protein and the solvent.
2.3. The Protein’Folding Problem
The ”central dogma” of molecular biology states that the flow of sequential information from nucleic acid to protein is unidirectional: nucleic acid sequences encode the sequence of proteins but once translation occurs, information cannot flow back from protein to nucleic acid. A possible extension of the central dogma would be to add that the sequence of the protein ”encodes” its three’dimensional structure. Indeed, this coding is sometimes called the ”second half of the genetic code.” Cracking this code would be equivalent to solving the ”protein folding problem.” Afinsen for the first time established that the information required to form native structure of the protein is encoded in its amino acid sequence (Afinsen, 1973). He demonstrated that denatured ribonuclease could refold spontaneously in vitro into its native three dimensional conformation without assistance from any cellular machinery. This suggested that the amino acid sequence of proteins has all the information required for native structure formation. Since then, prediction of the three dimensional structure of a protein from its amino acid sequence has become a major goal in the field of protein science. The ability of the linear polypeptide to select the most stable conformation spontaneously and usually quite rapidly from a myriad of alternatives has given rise to the protein folding problem. Native states of proteins correspond to the structures that are thermodynamically most stable under physiological conditions (Dobson et al., 1998). For a given protein, a large number of conformations are possible considering different _ and �� angles for different amino acid residues. With an average of m equally probable conformations per amino acid residue and n residues in the polypeptide chain, the total number of possible conformations of the polypeptide chain will be mn. Even if we consider that only a small fraction of these conformations are permitted still, the total number of possible conformations is so large that a systematic search for unique native structure would take an astronomical length of time. However, proteins are observed to fold on seconds to minutes time scale. This argument is commonly known as Levinthal’s paradox (Levinthal, 1968). The paradox could, however disappear if we argue that there is a conformational bias in the sequence towards the native structure. Such an argument can also explain Anfinsen’s observation. Thus, protein folding is not that random a process and must be following a well guided path to attain the native structure. The concept of folding pathways motivated a large number of experimental studies aimed at finding the specific ”folding intermediates” and also gave rise to a number of models describing the folding process. For example, the nucleation/growth model (Wetlaufer, 1973) tried to resolve Levinthal’s paradox by assuming that the rate’limiting step of the folding process is a nucleation event, presumably the formation of smaller structural units, and once nucleation occurs the nuclei grow fast and the folding process rapidly completes. This model is not consistent with the large number of observations where folding intermediates were observed. According to the ”diffusion’collision’adhesion model” (Karplus and Weaver, 1976), fluctuating microdomains (portions of secondary structure or hydrophobic clusters) move diffusively and repeatedly collide with each other. Collisions can lead to a coalescence of the microdomains into larger units (adhesion). The rate’limiting stage is assumed to be the diffusion process. This model is well supported by many experiments (Karplus and Weaver, 1994). The ”framework model” (Baldwin, 1989) states that the folding process is hierarchical, starting with the formation of the secondary structure elements, and the docking of the preformed substructures is the rate’limiting step. The ”hydrophobic collapse model” (Dill, 1985) is based on the view that the hydrophobic effect is the main driving force of folding, and the process starts with a rapid collapse of the chain, followed by the formation of the secondary structure. In fact, whether hydrophobic collapse or secondary structure formation occurs first has remained a largely undecided issue even to this day. Finally, the ”jigsaw puzzle model” (Harrison and Durbin, 1985) denied the necessity of a unique, directed folding pathway and stated that each protein molecule can follow a different route to the native structure, just like there are multiple ways to solve a jigsaw puzzle. This idea is actually consistent with a ”new view” of protein folding, which gained popularity in the 1990s: the energy landscape view. The energy landscape view likens the energy landscape of a protein to a funnel, with the native structure at its global minimum, and each molecule may follow a different microscopic route from the top to the bottom (Figure 2.2.). The many models of protein folding are not mutually exclusive; they try to grasp different aspects of folding, and experimental results give some support to each model we mentioned. A newer model, named ”nucleation’condensation model,” is an attempt to unite the features of both the framework and the hydrophobic collapse mechanisms (Fersht, 1995; Fersht, 1997). In this model, long’range and other native hydrophobic interactions form in the transition state to stabilize the otherwise weak secondary structure. The framework and the hydrophobic collapse models are viewed as two extremes of the nucleation’condensation mechanism; most proteins fold by a mechanism that is somewhere between the two extremes, i.e., secondary structure and hydrophobic interactions form nearly simultaneously and synergistically (Daggett and Fersht, 2003).
Figure’ Schematic representation of a funnel’shaped energy landscape. The width of the funnel represents the conformational freedom of the chain. The vertical axis represents the free energy; as free energy decreases, the nativeness of the chain increases. Denatured (unfolded) states are at the top of the funnel while the native state is the global minimum. There is some ruggedness in the energy landscape near the native state.
2.4. In vivo vs in vitro protein folding process
However, it has been shown that in the living cells, protein factors are involved in the folding of the polypeptides, which are referred to as molecular chaperones (Seckler and Jaenicke, 1992; Walter and Buchner, 2002). As many of them have been discovered in correlation with the heat shock response, they are also called heat shock proteins or Hsps (Lindquist and Craig, 1988). Molecular chaperones are a group of proteins related in their function. They do not provide any additional steric information for the folding of the target protein, their role is to suppress nonproductive interactions, and in that way help the protein to acquire its native conformation (Hartl et al., 2011; Seckler and Jaenicke, 1992; Walter and Buchner, 2002) Most chaperones can be defined by common functional features. In the first place, it is their ability to bind hydrophobic patches of unfolded, misfolded or partially folded polypeptides, and thus prevent aggregation process. Along with molecular chaperones, foldases play an important role in protein folding, as they
accelerate potentially slow steps in the folding process and thereby prevent aggregation.
There are two types of foldases, with different modes of action i.e. peptidyl-prolyl cis or trans isomerase (PPI), which catalyzes isomerization of prolyl peptide bonds; and protein-disulfide isomerase (PDI), which catalyzes formation and isomerization of disulfide bonds for proper folding (Gupta and Tuteja, 2011). Protein folding is carried out in the highly crowded environment of the cell, at high protein concentration, with many proteins being synthesized simultaneously (Hartl and Hayer-Hartl, 2009). Under such conditions misfolding and aggregation seem to be unavoidable. The main difference between protein folding in vitro and in vivo is that in the cell, this process is regulated and facilitated by the network of molecular chaperones and folding helpers. In contrast to in vivo protein folding, where all proteins have to gain their functional structure under the same conditions, folding conditions in vitro have to be determined for each protein empirically. Optimal in vitro folding conditions are defined by the specific characteristics of each particular protein and involve such parameters as protein concentration, temperature, incubation time and refolding buffer (Lange and Rudolph, 2005). Additionally, under in vivo conditions slow rate limiting reactions in the folding and association pathway are influenced by molecular chaperones and folding helpers, which prevent non-productive side reaction leading to misfolded protein and aggregation (Buchner and Rudolph, 1991; Kiefhaber et al., 1991). Renaturation process can be initiated by the decrease of the high concentrations of the denaturant and reducing agents, used for denaturation. Aggregation of unfolded protein and misfolding of folding intermediates compete with the refolding reaction, which is a major complication during the renaturation process
2.5. Disulfide bonds and protein folding
For many recombinant proteins, the formation of correct disulfide bonds is vital for attaining their biologically active three- dimensional conformation. The formation of erroneous disulfide bonds can lead to protein misfolding and aggregation into inclusion bodies. The process by which a protein attains its native disulfide bonds and its native structure is named oxidative folding. Pairing between cysteins is a complex process where oxidation, reduction and disulfide reshuffling might compete inside the cells. In eukaryotic cells, disulfide bond formation occurs in the endoplasmic reticulum before the transport of most extracellular and membrane bound proteins. Intracellular disulfide containing proteins are rarer because the cytocol tends to be a reducing environment. Disulfide formation in vivo is catalyzed by specialized enzymes, such as protein disulfide isomerase (PDI), which catalyzes internal disulfide bridges.
Disulfide bridges provoke well-known chemical and structural changes, allowing us to use them as good reporters of folding pathways. In oxidative folding experiments in vitro, the protein is placed under denaturing and reducing conditions. Then, the sequential disulfide- bond formation is promoted in removing both chaotropic and reducing agents. Monitoring disulfide regeneration can be performed in the absence and presence of redox agents (Chatrenet and Chang, 1993). Oxidative and reductive reagents increase the efficiency of the oxidative folding process, and promote disulfide reshuffling allows intermediates to attain the native disulfide connectivity. Different redox conditions can be used to modify refolding refolding rates and folding efficiencies, remaining the folding pathways unaffected (Chang, 1994)
2.6. Thermodynamics Of Protein Folding
A unified view of protein folding should be general enough to interpret the diverse experimental findings of the field. Thermodynamics offers such a universal approach. Thermodynamic systems in equilibrium occupy the states with lowest Gibbs free energy at constant pressure and temperature. The Gibbs free energy (G) consists of an enthalpy and an entropic term
��G(q) =�� H(q) ‘ T(q)��S
where H is the enthalpy, T the absolute temperature, and S the entropy of the protein, and q represents the reaction coordinate used to describe the progress of the protein advancing from the unfolded toward the native state. Under physiological conditions, proteins maintain their native structure because the favorable enthalpic term arising from the solvent and protein interactions exceeds in magnitude the unfavorable entropic term, and therefore the native state has a smaller Gibbs free energy than the denatured state. The stability of the protein depends on the solvent’solvent, protein’solvent, and protein’protein interactions. These interactions depend on the intensive parameters that describe the thermodynamic state of the system.
The enthalpic and the entropic terms are large, but of opposite sign, and almost cancel each other. The Gibbs free’energy difference between the biologically active and denatured states of the proteins is rather small (Scharnagl et al., 2005). Proteins are stable only within a narrow range of conditions and can be denatured by changing virtually any of the intensive parameters (Shortle, 1996). Experiments prove that proteins can be unfolded by heat (Tsai et al., 2002; Prabhu and Sharp, 2005), cold (Franks, 1995; Kunugi
and Tanaka, 2002), high pressure (Smeller, 2002; Meersman et al., 2006), extreme pH (Puett, 1973; Fitch et al., 2006), and addition of salts (Pfeil, 1981). Studies of protein stability and folding systematically change one or more of the intensive parameters and follow the shift of equilibrium. There is a broad selection of methods that can be used to follow the structural changes of the proteins, including fluorescence (Isenman et al., 1979; Vanhove et al., 1998), circular dichroism (Kelly and Price, 2000), nuclear magnetic resonance (Englander and Mayne, 1992; Kamatari et al., 2004), and mass spectroscopy (Miranker et al., 1996; Konermann and Simmons, 2003) and many more.
Several thermodynamic coordinates have been used to describe the ”nativeness” of a given protein state. Thermodynamic reaction coordinates use a thermodynamic parameter, e.g., Gibbs free energy and/or entropy, to define the distance between the native state and the actual state of a protein. An important thermodynamic reaction coordinate often used to describe the folding process is the number of native contacts present in the conformation, which proved useful in interpreting simple folding processes. The Gibbs free’energy barrier to folding is determined by the unfavorable loss in configurational entropy upon folding and the gain in stabilizing native interactions. Starting from the unfolded protein, the polypeptide chain has to fold partially in order to bring together the residues that need to form the contacts stabilizing the native structure. The constrained polypeptide chain has smaller entropy, which means higher Gibbs free energy. As native contacts form, the enthalpy term decreases, the protein is stabilized. The rate’limiting step in the folding process is the formation of the transition state, i.e., the conformation that has the highest Gibbs free energy on the folding pathway (Chan and Dill, 1998; Lindorff’ Larsen et al., 2005). The simplest model for unfolding and refolding involves a single cooperative folding step, in which the unfolded (U) and folded (F) states of the protein interconvert: U ‘ F. This simple mechanism well describes the folding of several small proteins (Gillespie and Plaxco, 2004). The formation of a contact between two residues in the transition state involves an entropic cost which depends on the sequence separation of the two residues: the longer the chain between them the greater the entropic cost, and this entropic cost contributes to the height of the Gibbs free’energy barrier between the unfolded and the folded state.
Intermediate structures were observed to accumulate during the folding of many proteins (Englander, 2000). Such intermediate states are trapped structures that have low Gibbs free energy. Presence of such intermediates describes multi- state protein folding process.
2.7. Protein stability
The most common method for the reliable estimation of protein stability is the determination of equilibrium constant between the native (N) and the unfolded protein (U), which is of the order of 105 or higher for a typical protein. This means that there is one unfolded molecule in 0.1 million molecules, which makes reliable experimental measurement of equilibrium constant difficult. In order to overcome this, equilibrium between U and N can be perturbed such that the fraction of U or N is populated significantly for reliable estimations. Chemical denaturants like urea and GdnHCl are usually employed for such studies. These chemicals denature proteins by solvating non polar groups better than water does. They solubilize all constituent parts of a protein from its polypeptide backbone to its hydrophobic side chains (Dill, 1990). This approach allows one to measure protein stability under perturbed conditions. Stability of protein under unperturbed conditions can be obtained by extrapolating the measured values under perturbed conditions back to the unperturbed conditions. Measurements of this type require an understanding of the mathematical relation that describes the dependence of protein stability on denaturant concentration. Such a relation is given by linear extrapolation model (LEM) which states that free energy of transfer of the side chains and polypeptide backbone from water to denaturant is linearly proportional to the concentration of denaturant (Tanford, 1968; Tanford, 1970 and Greene and Pace, 1974). Thus, the free energy of denaturation at any particular concentration of denaturant is given by
��GU-N = ��GH2O U-N ‘m U-N [denaturant]
Where, ��GH2O U-N is the value in water that is obtained by extrapolation to zero denaturant concentration and mU-N is the dependence of free energy change of unfolding on denaturant concentration and has the dimensions of cal/mol/M. It is defined as the change in surface area of upon unfolding and thus, large proteins have large m values as compared to small proteins (Myers et al., 1995). The free energy equation is valid only for a reversible reaction, i.e. where N’U. At equilibrium, ��GU-N is zero because there are equal concentrations of U and N and ��GU-N = -RT ln ([U]/[N]). The equation for ��GU-N may be rearranged to obtain,
[U]/[N] + [U] = exp({m U-N[denaturant] – ��GH2O U-N}RT)/ 1 + exp({mU-N[denaturant] – ��GH2O U-N}RT)
The fraction unfolded, [U]/[N] + [U], may be determined by spectroscopy (fluorescence, circular dichroism or absorbance):
FU = (X-XN) / (XU – XN)
where, FU is the fraction unfolded; X is observed spectroscopic signal; XN is spectroscopic signal of native protein; XU is the spectroscopic signal of the unfolded state (The equations are adapted from Fersht, 1999).
The above equations apply to a simple two state equilibrium of N’D where protein denaturation is a cooperative (all or none) transition in which denaturation occurs in a single step without any intermediates accumulating. If an intermediate species has significant stability relative to native or unfolded species, it will accumulate at equilibrium and in such situations appropriate equations should be used (Rumfeldt et al., 2008 and Walters et al., 2009). Thus, thermodynamics of large and multi-domain proteins becomes complex to analyze due to occurrence of multistate transitions.
2.8. Importance of thermodynamic studies
Apart from gaining insight into the fundamentals of the folding process, thermodynamics studies are very important for several medicinal and industrial interests. Knowing that proteins can perform a diverse set of functions, researchers are interested in designing novel protein molecules that can perform a function of interest. In order to carry out such studies, the kinetic accessibility of the native state and its stability needs to be predicted from its amino acid sequence. During the process of protein folding, many proteins tend to misfold (Galani et al., 2002 and Gianni et al., 2010). These misfolded structures ultimately give rise to protein aggregates and inclusion bodies (Prusiner, 1998; Galani et al., 2002 and Andrews and Roberts, 2007). Protein aggregation is the main cause of several neurodegenerative diseases (Dobson, 2003 and Chiti and Dobson, 2006) while inclusion body formation is a major hurdle in the large scale production of purified proteins in industries. The knowledge of equilibrium protein folding process could be used to prevent protein misfolding which could prove quite helpful in tackling medicinal and industrial issues. Elucidation of thermodynamics of protein aggregation and disaggregation processes will be a key to understand a variety of neurodegenerative diseases. By comparing thermodynamic parameters of a protein from various origins may provide close insight to structure – function relationship of the protein.
2.9. Experimental techniques involved
Many different experimental techniques are applied to study the characteristics and properties of proteins. It involves over- expression of recombinant protein in E. coli and further enhancement of expression using various chemical chaperones (osmolytes), purification of recombinant protein for in vitro studies. Many more experimental techniques can also be utilized to test and gain insight into the stability and reversibility (folding) of proteins when subjected to different environmental conditions, such as changes in temperature, pH and solution medium. Below is a general introduction to the experimental techniques utilized often in our research: enzyme assay, fluorescence spectroscopy and far UV ‘ CD.
2.9.1. Recombinant protein expression in E. coli
Recombinant DNA (rDNA) technology is an essential and necessary tool for the large scale production of proteins of industrial, therapeutic and research significance. The combination of rDNA technology and the scale-up processes provide a platform for large-scale production of recombinant proteins to satisfy the ever-increasing demand. The pre-requisite for the structural analysis and biopharmaceutical research purposes is that, the recombinant protein used must be in the native conformation. Due to the recent developments in the rDNA technology invariably, gene of any length and of any origin/source can be expressed in any of the expression systems like animals, plants and microorganisms. Each of the host organisms possesses intricate characteristics which favours efficient expression of recombinant products.
The enteric bacterium, E. coli is the most widely used prokaryotic heterologous expression system. In order to achieve high yields of the recombinant proteins, the regulations of the host transcriptional and translational machineries should be considered (Makrides, 1996; Chen, 2012). E.coli which has been the workhouse of genetic engineers both for basic studies and expression of recombinant proteins because of well-studied and well characterized genome, easy genetic manipulation, cost effectiveness due to inexpensive culturing, fast and high expression, recovery of high yield of recombinant proteins, tolerance for majority of foreign proteins etc. All these characteristics make it a very good expression system and favors recombinant protein production.
Even though, E.coli expression system exhibits some limitations like formation of inclusion bodies, absence of post-translational modification machinery, presence of endotoxins in the cellular extracts of therapeutic proteins; its utility is inevitable in the recombinant protein production.
2.9.2. Enhancement of expression of recombinant protein by Osmolytes
When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species.
Osmolytes are naturally occurring organic compounds, which represent different chemical classes including amino acids, methylamines, and polyols (Table 2.1). By accumulating high concentrations of osmolytes, organisms adapt to perturbations that can cause structural changes in their cellular proteins. Osmolytes shift equilibrium toward natively-folded conformations by raising the free energy of the unfolded state. As osmolytes predominantly affect the protein backbone, the balance between osmolyte’backbone interactions and amino acid side chain’solvent interactions determines protein folding.
Abnormal cell volume regulation significantly contributes to the pathophysiology of several disorders, and cells respond to these changes by importing, exporting, or synthesizing osmolytes to maintain volume homeostasis. In recent years, it has become quite evident that cells regulate many biological processes such as protein folding, protein disaggregation, and protein’protein interactions via accumulation of specific osmolytes. Many genetic diseases are attributed to the problems associated with protein misfolding/aggregation, and it has been shown that certain osmolytes can protect these proteins from misfolding. Thus, osmolytes can be utilized as therapeutic targets for such diseases (Naturally Occurring Organic Osmolytes: From Cell Physiology to
Disease Prevention’..Shagufta H. Khan1, Nihal Ahmad2, Faizan Ahmad3, and Raj Kumar1).
Table 2.1. Various classes of osmolytes used for the checking of overexpression in zDHFR
S.no. Osmotic Class Name Structure Molecular weight
(gm/l)
1. Amino acids and their derivatives Proline 115.13
Glycine 75.06
2. Carbohydrates Sorbitol 182.17
Half saccharide Glycerol 92.09
Monosaccharide D-Glucose 180.16
Disaccharide Sucrose 342.30
3. Methylammonium and methylsulfonium solutes Glycine betain 117.14
4. Carbamides Urea 60.06
5. Salts Sodium Chloride NaCl 58.44
In general, adding solutes such as osmolytes and macromolecules (Knoll, D.; Hermans, J. J Biol Chem 1983, 258, 5710’5715; Hermans, J. J Chem Phys 1982, 77, 2193’2203) stabilizes proteins and alters the affinities of proteins for one another. At the solute concentrations required to observe these effects, there is a significant decrease in water concentration and an increase in the volume occupancy. While changes in both solute and water concentrations and in volume fraction undoubtedly contribute to changes in the free energy of the protein, the molecular-level interpretations of solute- induced changes in protein stability generally focus on only one of these effects
(Osmolyte-Induced Changes in Protein Conformational Equilibria Aleister J. Saunders1,*
Paula R. Davis-Searles2 Devon L. Allen2 Gary J. Pielak1,2 Dorothy A. Erie2).
Figure 3. A possible therapeutic mechanism of use of osmolytes to protect misfolded/aggregation-prone proteins in disease conditions. Under pathological conditions, the structure of natively-folded functional protein (PF) is compromised because of partial folding (PPF) that could either result in unwanted degradation or into formation of soluble precursors that forms amyloids. Osmolytes can help in converting (PPF) back into (PF) conformation and thereby restoring proper functions of protein
leading to prevention of disease.
Various osmolytes have been used to prevent aggregation and to stabilize protein molecule for example addition of proline or glycine in concentrations of up to 1 M to the refolding buffer has also been reported to suppress aggregation and improve refolding efficiencies (Han et al., 2010; Ito et al., 2008; Kim et al., 2006a; Meng et al., 2001; Samuel et al., 2000). Polyol osmolytes are also very popular osmolytes used for stabilization of aggregation prone proteins. Polyols are alcohols with the number of hydroxyl groups. Addition of polyols was shown to result in improved refolding and stabilized protein structure (Cleland et al., 1992; Dworeck et al., 2011; Kim et al., 2006a; Mishra et al., 2005). Within this group, glycerol is probably most frequently applied to enhance the refolding efficiency, and is
usually applied in a range of 10 – 50% v/v (Mishra et al., 2005; Tieman et al., 2001; Vagenende et al., 2009; Wang et al., 2009). It was proposed that glycerol prevents protein
aggregation by inhibiting protein unfolding and by stabilizing the aggregation-prone
intermediates through preferential interactions with hydrophobic surface regions that favor amphiphilic interface orientations of glycerol (Vagenende et al., 2009). Also, there are many reports suggesting the in- vivo role of osmolytes in correcting the folding defects of proteins for example, glycerol can correct the temperature sensitive folding defect of the human cystic fibrosis transmembrane conductance regulator mutant protein and tumor suppressor protein in cells (Sharma et. al., 2012) jpp
Polyols such as sorbitol was reported to positively influence the refolding process (Majumder et al., 2001; Mishra et al., 2005; Yu and Li, 2003). Some denaturants also reported to be used as additives to enhance renaturation. They are used at nondenaturing concentrations for example GdmCl and urea . The positive impact of these substances can be explained by their denaturing characteristics, as they may stabilize intermediates and unfolded protein, thus increasing their solubility and decreasing aggregation.
In the present study, we have tried to enhance the in-vivo expression of zebrafish DHFR in E.coli cells by investigating the effect of various osmolyte supplementation in the culture media on the production of functional zDHFR protein.
2.9.3. Purification of recombinant protein by affinity chromatography
The first challenge which comes into existence duting protein folding study is to isolate and to purify the protein of interest from the producing organism in enough quantity for in vitro studies. Generally preferred technique is affinity chromatography. It makes use of a specific and reversible binding of the target molecule to the affinity ligand, coupled to an inert chromatography matrix. IMAC (immobilized metal affinity chromatography) is a typical example, it is based on the formation of chelate complex between certain protein residues (histidines, cysteins and to some extent tryptophanes) and matrix-bound transition metal cations like Ni2+, Co2+, Zn2+ and Cu2+. IMAC is used for the purification of recombinant proteins carrying a polyhistidine tag, which is a sequence of usually 6 or 10 histidines on either N- or C- terminal of the target protein. Due to its characteristics in terms of selectivity and loading capacity Ni2+ is the most often used ion. The his-tag binds to nickel ions with micromolar affinity. The target protein is usually eluted with 100-500 mM Imidazole, which competes with the histidine-tag for the binding ligands, however, low concentrations of imidazole (5-20mM) in the washing buffer contribute to the purification efficiency, as all interactions of unspecifically bound proteins with the metal ion are abolished. The choice of pH of the chromatography buffer is also of great importance, as at low pH ( or =2 M urea or at temperatures of 65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.
J Mol Biol. 2002 Jan 11;315(2):193-211.
Human DHFR case studies
Highly divergent dihydrofolate reductases conserve complex folding mechanisms.
Wallace LA1, Robert Matthews C.
Author information
Abstract
To test the hypothesis that protein folding mechanisms are better conserved than amino acid sequences, the mechanisms for dihydrofolate reductases (DHFR) from human (hs), Escherichia coli (ec) and Lactobacillus casei (lc) were elucidated and compared using intrinsic Trp fluorescence and fluorescence-detected 8-anilino-1-naphthalenesulfonate (ANS) binding. The development of the native state was monitored using either methotrexate (absorbance at 380 nm) or NADPH (extrinsic fluorescence) binding. All three homologs displayed complex unfolding and refolding kinetic mechanisms that involved partially folded states and multiple energy barriers. Although the pairwise sequence identities are less than 30 %, folding to the native state occurs via parallel folding channels and involves two types of on-pathway kinetic intermediates for all three homologs. The first ensemble of kinetic intermediates, detected within a few milliseconds, has significant secondary structure and exposed hydrophobic cores. The second ensemble is obligatory and has native-like side-chain packing in a hydrophobic core; however, these intermediates are unable to bind active-site ligands. The formation of the ensemble of native states occurs via three channels for hsDHFR, and four channels for lcDHFR and ecDHFR. The binding of active-site ligands (methotrexate and NADPH) accompanies the rate-limiting formation of the native ensemble. The conservation of the fast, intermediate and slow-folding events for this complex alpha/beta motif provides convincing evidence for the hypothesis that evolutionarily related proteins achieve the same fold via similar pathways.
Protein Sci. 1996 Dec;5(12):2506-13.
Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.
Gross M1, Robinson CV, Mayhew M, Hartl FU, Radford SE.
Author information
Abstract
An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by completeunfolding of this protein substrate on the chaperonin surface.
CHAPTER 3
MATERIALS AND METHODS
3. Materials and methods
Methods are adopted from standard methods developed at Molecular Biophysics lab, Amity Institute of Biotechnology, Amity University, Noida, India and Kusuma School of Biosciences, Indian Institute of Technology, New Delhi, India.
3.1. Bacterial vectors
DH5��, BL21 (DE3) and BL21 (DE3) Rosetta E. coli strains were used for the expression and purification of DHFR from three different sources (E. coli, human and zebrafish). pET16b plasmid expressing E. coli DHFR gene was a generous gift from Prof. Taguchi, Tokyo Institute of Technology, Japan. pET43.1a plasmid containing human DHFR gene and zebrafish DHFR gene was a generous gift from Prof. Tzu- Fun Fu, National Cheng Kung University, Taiwan. The list of recombinant protein systems used in the present study is given in Table 3.1.
Table 3.1. List of recombinant vectors used
Name Vector Selection Marker E. coli expression system
E. coli DHFR pET16b AmpR BL21 (DE3)
Human DHFR pET43.1a AmpR BL21 (DE3) Rosetta
Zebrafish DHFR pET43.1a AmpR BL21 (DE3) Rosetta
3.2. Molecular biology methods
3.2.1. Cultivation and storage of E. coli
E. coli was cultivated either in Luria-Bertani (LB) broth medium or on the LB plates, containing appropriate antibiotics, in a thermostated incubator at 37��C. The growth of bacterial cells in liquid cultures was monitored by measuring OD at 600 nm (where, OD600nm = 1 corresponding to approx. 8×108 cells/ml) (Sambrook J and Maniatis T, 1989). For the long term storage 5 ml of cells in exponential growth phase, were centrifuged for 3 min at 5000 x g at 4�� C, sediment was resuspended in 600 ��l LB medium, subsequently 400��l of 50 % glycerol were added. Cultures were stored at ‘ 80��C.
3.2.2. Preparation of competent E. coli cells
(Sambrook J and Maniatis T, 1989)
Respective E. coli cells were inoculated in 50 ml LB medium and incubated at 37��C till OD600 of 0.4- 0.6 was reached. The cells were kept at 0�� C for 10 min and recovered by centrifugation at 4000 rpm for 10 min at 4�� C. Cell pellet was resuspended in 20 ml of ice-cold sterile CaCl 2 and kept in ice for 30 min. Competent cells were finally recovered by centrifugation at 4000 rpm for 5 min at 4�� C . 20 % glycerol stock of competent cells were prepared and stored at -80�� C.
3.2.3. Transformation of competent E. coli cells and cultivation
200 ��L of competent E. coli cells were mixed with 40 ng of plasmid DNA, and incubated on ice for at least 30 min, then transferred for 90 sec to 42��C for a quick heat shock step, and cooled down on ice for another 5 min. After the addition of 1ml LB, cells were gently shaken at 37��C for at least 1 h, which enabled transcription of resistance genes and synthesis of the antibiotics processing enzymes. Subsequently, cells were centrifuged at 4000x g for 5 min and either plated or inoculated in to the liquid media, with the appropriate concentration of antibiotic. E. coli cultures were incubated overnight at 37��C.
3.2.4. Amplification of plasmid DNA
For amplification of plasmid DNA, single colonies were transferred into 5 ml of LB containing
the appropriate antibiotic and grown in a rotary shaker at 37��C overnight. 1.5ml of the culture was centrifuged in an Eppendorf tube and the pellet was subjected for plasmid isolation by alkaline lysis method (Sambrook and Maniatis, 1989). This protocol was followed for plasmid isolation of all the three recombinant DHFR proteins.
3.3. Protein chemical methods
3.3.1. Protein purification by affinity chromatography
The following chromatographic method was applied for protein purification during this work. The purification was confirmed by 12 % SDS- PAGE after every step. Affinity chromatography makes use of a specific and reversible binding of the target molecule
to the affinity ligand, coupled to an inert chromatography matrix. IMAC (immobilized metal affinity chromatography) is a typical example, it is based on the formation of chelate complex between certain protein residues (histidines, cysteins and to some extent tryptophanes) and matrix-bound transition metal cations like Ni2+, Co2+, Zn2+ and Cu2+. IMAC is used for the purification of recombinant proteins carrying a polyhistidine tag, latter is a sequence of usually 6 or 10 histidines on either N- or C- terminal of the target protein. Due to its characteristics in terms of selectivity and loading capacity Ni2+ is the most often used ion. The his-tag binds to nickel ions with micromolar affinity. The target protein is usually eluted with 100-500 mM Imidazole, which competes with the histidine-tag for the binding ligands, however, low concentrations of imidazole (5-20mM) in the washing buffer contribute to the purification efficiency, as all interactions of unspecifically bound proteins with the metal ion are abolished. The choice of pH of the chromatography buffer is also of great importance, as at low pH (http://www.expasy.org. Protein samples were measured in UV Quartz cuvettes, all spectra were buffer corrected.
3.5.2. Fluorescence spectroscopy
Fluorescence spectroscopy is a spectrochemical method of analysis where the molecules under study are excited at a certain wavelength and remain excited for 1- 10 nanosec. After conformational changes in the protein molecule due to the change in its environment, it emits radiation of a longer wavelength due to energy dissipation. Upon excitation by the light of an appropriate wavelength, the electronic state of the molecule changes from the ground to one of the excited states. The excited electronic state is usually the first excited singlet state. From the excited state relaxation to the ground state can occur via several processes. Fluorescence corresponds to the emission of light occurring due to the relaxation of the molecule from the singlet excited state to the singlet ground state. In this work fluorescence spectroscopy was applied to determine the activity of DHFR, the protein was excited at 295 nm, emission from 310- 400 nm was monitored.
3.5.3. Far UV- CD spectroscopy
Circular dichroism (CD) spectroscopy relies on the differences between the absorption of left-handed polarized light and right-handed polarized light which arise due to structural asymmetry of chromophore. Conformationally ordered structure results in positive and negative signals in CD spectrum whereas zero CD intensity can be obtained in case of randomly ordered structures. CD spectroscopy in the “far-UV” spectral region (190-250 nm) can be helpful in determining secondary structure of a protein. At this range of wavelength the chromophore is the peptide bond, which gives signal when it is present in a native, folded environment. Molar residue ellipticity (MRE; deg cm2 dmol-1) was calculated by using the following formula:
[��] = ��/n.c.l
where �� is measured ellipticity (millidegree), n is the number of amino acid residues in the protein, c is the molar concentration of protein, and l is the path length (mm). The values in millideg were converted to molar residue ellipticity with the help of the software associated with the instrument by using the concentration of the protein solution and the path length of the cuvette. This technique gives the percentages of the different secondary structures (��-helix, ��-sheet and random coil) estimated from the spectra.
3.6. Characterization of DHFR
3.6.1. Confirmation of Molecular mass by Mass Spectroscopy
The molecular weight of purified protein was verified by MALDI TOF mass spectrometry (Bruker). zDHFR has an extinction coefficient of 24075 M’1 cm’1 at 280 nm as computed using the ProtParam tool of ExPASy (http://web.expasy.org/protparam/). It was used for the determination of concentration of purified protein.
3.6.2. Estimation of disulphide bond
1ml protein solution (0.3%) in 0.1M sodium phosphate buffer, pH 7.4 containing SDS and 1mg EDTA was added 0.05ml of 0.01M DTNB solution in the same buffer under N2 atmosphere. Absorbance was observed after 30 minutes at 412nm. Concentration was measured from known value of molar extinction coefficient and expressed as moles of sulfhydryl groups per mole of the protein.
3.6.3. Enzyme activity
zDHFR activity was determined by the spectroscopic method using the following reaction scheme:
NADPH NADP+
DHF THF
DHFR
Where, DHF and THF are dihydrofolate and tetrahydrofolate, respectively. The assay depends on the reversible NADPHdependent reduction of dihydrofolic acid to tetrahydrofolic acid in the presence of DHFR . This reaction was monitored by the decrease in absorbance of NADPH at 340 nm. The control experiment was carried out in the absence of any DHFR protein to account for the blank contribution from NADPH. The value of blank was negligible as compared to the assay result in the presence of DHFR protein. The decrease in the concentration of NADPH in the presence of DHFR protein is thus attributed to the enzyme catalysed reaction only.
The decrease in NADPH concentration was monitored by measuring its absorbance at 340 nm at 25��C. The standard assay mixture for zDHFR was composed of assay buffer (20 mM Tris-HCl, 25 mM KCl, pH 7.4, 1 mM GSH, 0.1 mM GSSG and 10 % glycerol along with substrate,100 ��M dihydrofolic acid and co-factor,140 ��M NADPH. One unit of enzyme has been described as the amount of enzyme required to oxidize 1 ��mole of dihydrofolate per min, which is based on its molar extinction coefficient of 12,300 M’1 cm’1 at 340 nm. All enzyme assays were performed in triplicate and to minimize the degradation of substrate and cofactor, NADPH and DHF were prepared fresh for each experiment, incubated in ice and consumed within 2 h of experimentation.
3.6.4. Intrinsic tryptophan fluorescence Spectra
Steady state fluorescence spectra was recorded on a Perkin-Elmer luminescence spectrometer LS 55 (Perkin-Elmer, USA) at 25��C using an optical cuvette with path length of 1 cm. Intrinsic tryptophan fluorescence spectra of the purified zDHFR in 20 mM Tris pH 7.4, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG and 10% glycerol buffer was recorded after exciting the protein sample at 295 nm using excitation and emission slit widths of 5 nm and 10 nm, respectively. Emission spectra were recorded between 310-400 nm. Baseline corrections were perfoemed using buffer without protein in all cases. Final zDHFR concentration was 2 ��M.
For ANS fluorescence spectra, protein solution was incubated with 50 ��M ANS at room temperature for 5 min after which the sample was excited at 370 nm and ANS emission spectra were recorded in the 400-600 nm range. Free ANS gives maxima at 468 nm which is shifted to 468- 440 nm range when ANS binds to the protein. Here, excitation and emission slits used were 5 and 10 nm, respectively. Scan rate was 60nm/ min. Final DHFR concentration was 0.5 ��M.
3.6.5. Far-UV CD spectra
Far-UV CD spectra of purified zDHFR protein were recorded on JASCO J-815 CD polarimeter
(JASCO, Japan), flashed with nitrogen gas. The buffer used for the measurement was 20 mM Tris pH 7.4, 25 mM KCl. Protein concentration used was 5 ��M. The spectra were recorded between 200-250 nm in a 0.2 cm cuvette with a 2 nm band width and 1 s response time. Each spectrum represents average of three accumulations. All spectra were corrected for background signals by buffer alone. The percentages of the different secondary structures (��-helix, ��-sheet and random coil) were estimated from the spectra by the method of Yang et al., 1986.
3.7. In vivo folding of DHFR
In order to monitor the in vivo folding of DHFR, fractionation experiments were carried out. The principle behind these experiments is that the folded protein is soluble and physiologically active, whereas the misfolded and aggregated proteins are insoluble and have ideally no functional activity. Thus, when the E.coli cells containing the over-expressed proteins are lysed and centrifuged, folded and soluble proteins come in the supernatant and misfolded or aggregated stuff appears in the pellet. BL21 E.coli cells containing the desired recombinant plasmids were grown in 10 ml LB media at 37��C and induced with appropriate concentration of IPTG as described earlier. After 6 h of induction at 25 �� C, equal amount of cells (OD600 = 0.8) were centrifuged (6000 rpm for 15 min), pelleted and re-suspended in 1 ml of lysis buffer and incubated for 30 min at 4��C. The cells were then disintegrated on ice by sonication in Branson sonifier 250 (30 cycles of 10 s each with 1 min interval on ice, output control 3, duty cycles 40). Immense care was taken to avoid any frothing during sonication. After sonication, the lysate was centrifuged at 12,000 rpm for 40 min. Whole supernatant was carefully removed (total volume 500 ��l) and the pellet was resuspended in an equal amount (500 ��l) of buffer (20 mM sodium phosphate pH 7.4, 500 mM NaCl, 0.1 mg/ml DNase, 0.2 M MgCl2, 1 mM PMSF and 0.1 mg/ml lysozyme) so that both the supernatant and pellet fractions are normalized. 20 ��l of supernatant and pellet fraction was taken and dissolved in 20 ��l of 2X SDS loading dye and the samples were loaded on a 12% SDS-PAGE gel after boiling. The gels were stained with coomassie blue stain and analyzed for the determination of in vivo folding of DHFR.
3.8. In vitro unfolding studies of DHFR
3.8.1. Buffers and solutions
The native or the refolding buffer used in equilibrium experiments was 20 mM Tris pH 7.4, 25 mM KCl in case of E.coli and human DHFR while the buffer composition for zebrafish DHFR comprises of 20 mM Tris pH 7.4, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG and 10 % glycerol. The refolding buffer conditions were optimized to achieve complete unfolding reversibility and to avoid any hysteresis in the equilibrium unfolding transition curve. The unfolding buffer was the native buffer containing 3 M GdnHCl or 7 M urea. Urea solutions were always prepared fresh just before the experiments. The refractive indices of the stock solutions of GdnHCl and urea were determined using an Abbe 3L refractometer for measuring the concentration of the denaturant. The equation given by Pace, 1986 was used to calculate the GdnHCl concentration
C= 57.177 (��N) + 38.68 (��N)2 + 91.6 (��N)3 + ”’..
Where, ��N is the difference between refractive index of the native buffer and GdnHCl containing native buffer. All measurements were carried out at room temperature (25��C).
Table 3.5
Origin of DHFR Refolding Buffer pH
E.coli 20 mM Tris HCl, 25 mM KCl 7.4
Human 20 mM Tris HCl, 25 mM KCl 7.4
Zebrafish 20 mM Tris HCl, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG, 10% glycerol 7.4
3.8.2. Chemical denaturant vs activity titration of DHFR
3.8.2.1. Dihydrofolate reductase (DHFR)
DHFR, is an oxidoreductase enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH as electron donor. To determine activity of DHFR, reduction of dihydrofolate was monitored at 340 nm (NADPH uptake). To monitor how enzymatic activity of DHFR changes upon denaturation, 0.2 ��M native zDHFR in above mentioned refolding buffer (Table 3.5) containing different concentrations of GdnHCl (0-3 M) and urea (0-7 M) was denatured for 2 and 3 h respectively, at 25��C. Following this, the activity of each of the protein solution was recorded using the activity assay procedure of DHFR as described before. GdnHCl concentration was kept same in the activity assay buffer as was during unfolding to avoid any refolding during the activity assay.
3.8.3. Equilibrium unfolding studies of zDHFR monitored by various biophysical tools
3.8.3.1. Intrinsic tryptophan fluorescence
2��M native zDHFR was denatured in different concentrations of GdnHCl (0-3 M) and urea (0- 7 M) for 2 and 3 h respectively at 25 ��C. Intrinsic tryptophan fluorescence spectra were recorded as described earlier.
3.8.3.2. ANS fluorescence
0.5 ��M native zDHFR was denatured in different concentrations of GdnHCl (0-5 M) and urea (0-7 M) for 2 and 3 h respectively, at 25��C. For recording ANS fluorescence, previously described protocol was followed. The optimized ANS concentration (50 ��M) was determined by measuring the minimum ANS concentration that produces maximum relative ANS fluorescence intensity.
3.8.3.3. Far UV-CD
Equilibrium unfolding of zDHFR monitored by far-UV CD at 222 nm was performed using optical cuvette with a 1 mm path length and the signal was averaged for 30 s. Relative millidegree values were calculated for each GdnHCl concentration as described earlier.
3.8.4. Equilibrium unfolding experiments in presence of various denaturants
Equilibrium unfolding of zDHFR under the influence of GdnHCl, Urea and acid at pH 2 was monitored by various biochemical and biophysical tools like loss of enzyme activity, changes in intrinsic tryptophan fluorescence and far-UV CD. Native DHFR (0.2 ��M for enzyme activity, 2 ��M for intrinsic tryptophan fluorescence, 0.5 ��M for ANS bound fluorescence and 5 ��M for far-UV CD) was incubated in presence of denaturant. Equilibrium unfolding was monitored as described earlier. Refolding conditions were optimized to achieve maximum refolding yield as assessed by loss of enzymatic activity in presence of chemical denaturants like Gdn HCl and urea. Buffer solutions were filtered through a 0.22-��m syringe filter before use. Each sample was corrected for blank contributions from buffer solutions in absence of protein with increased concentration of denaturant. (TT Kao et al, 2008) All samples were allowed to equilibrate in presence of denaturant at 25��C for 2 h for Gdn HCl denatured samples and for 3 h for urea and acid denatured samples.
3.8.5. Equilibrium Unfolding Studies of E.coli and human DHFR in presence of osmolytes monitored by various biochemical and biophysical approaches
3.8.5.1. Enzymatic Assay
Enzymatic activity of E.coli / Human DHFR samples in presence of various osmolytes such as 30% glycerol, 1M sucrose, 100 mM proline with final protein concentration 0.2 ��M containing different concentrations of GdnHCl (0-3 M) in 20 mM Tris-HCl, 25 mM KCl buffer, pH 7.4 was measured according to the method previously described. The enzyme activity of DHFR was then analyzed against different concentrations of GdnHCl.
3.8.5.2. Intrinsic Tryptophan Fluorescence Spectroscopy
Intrinsic fluorescence measurements of E.coli and human DHFR proteins in the presence of various osmolytes, like 30% glycerol, 1M sucrose, 100 mM proline were performed using Perkin-Elmer LS 55 spectrofluorimeter using an optical cuvette of 1 cm path-length. Samples were prepared by incubating DHFR proteins with final concentration of 1 ��M in standard unfolding buffer containing different concentrations of GdnHCl (0-3 M) at 25��C for 2 h. The samples were excited at 295 nm and emission spectra were recorded between 310-400 nm with excitation and emission slit width 5 nm and 10 nm, respectively(18). Percentage of denaturation was calculated from the relative fluorescence intensity data of the enzyme considering the intensity of the native protein is 0 % denaturation and the maximum changed value of the fluorescence intensity is 100% denaturation. The percentage of denaturation data were plotted against the concentration of GdnHCl to obtain the unfolding transition curve of E.coli and human DHFR.
3.8.5.3. Extrinsic Fluorescence Spectroscopy
Extrinsic fluorescence measurements of E.coli and human DHFR proteins in the presence of various osmolytes, like 30% glycerol, 1M sucrose, 100 mM proline were performed using Perkin-Elmer LS 55 spectrofluorimeter using an optical cuvette of 1 cm path-length. Samples were prepared by incubating DHFR proteins in the standard unfolding buffer, pH 7.4 for 2 h at 25��C. For both the cases, excitation wavelength was 370 nm and emission was recorded from 410-600 nm with excitation and emission slit width 5 nm and 10 nm, respectively (18). Emission readings were corrected for blank contributions from Gdn HCl at each concentration. Final ANS concentration in all cases was 50 ��M (18). Relative fluorescence intensity values of protein bound ANS was plotted against concentration of GdnHCl for E.coli and human DHFR.
3.9. In-vitro refolding of Zebrafish DHFR
As the refolding studies using E.coli and human DHFR were already reported, therefore the present study focusses on refolding of unexplored variant of Zebrafish DHFR.
3.9.1. Refolding in solution
For each refolding experiment, a total 200 ��M denatured protein were diluted 100-fold into the
respective refolding buffer so that the final GdnHCl concentration was below 0.1 M. Renaturation was carried out by series of dilution by Gdn HCl denatured protein in the refolding buffer such that the final protein concentration shall remain 0.2��M for enzymatic activity assay and 2��M for intrinsic tryptophan fluorescence spectroscopic analysis. Refolded samples were mixed by vortexing and incubated at 25�� C for 2 h. The yield of refolded protein was determined by quick centrifugation at high speed to eliminate the possibility of aggregates and monitoring through regain of enzymatic activity and change in intrinsic fluorescence. For reference, samples in triplicate for native DHFR protein in the respective buffer were prepared and incubated at 25�� C for 2 h.
3.10. Thermodynamic calculations
Thermodynamic stability of a protein is defined as the change in the unfolding free energy of the native protein (equation 2). The equilibrium between native and unfolded proteins, can be established by destabilizing the native state so that a detectable amout of population is present as the unfolded species, which is commonly performed by changing the environment of protein by increasing temperature or adding denaturants like urea or GdnHCl. In the present study, the unfolding free energy was calculated by using GdnHCl denaturation. Experimental data were analyzed using Sigmaplot (Systat software). To calculate thermodynamic parameters of Zebrafish DHFR, three state fitting was performed, in which the transition curves measured at 340 nm were fitted simultaneously for absorbance and fluorescence spectroscopy data, with the assumption that unfolding transition of this unexplored variant of DHFR comprise of three states only which are native (N), intermediate (I) and unfolded (U). The best fit values of the thermodynamic parameters were obtained by the method of non-linear least squares [Finn et al. 1992] and are summarized in Table 1. According to the results obtained, we were able to assume a three-state mechanism of unfolding in which a stable intermediate state (I) is populated during the equilibrium unfolding transition of Zebrafish DHFR from N to U. The unfolding reaction is thus represented by the following scheme:
KNI KIU
N I U
KNU
Where N, I and U represent the native, intermediate and unfolded form of zDHFR respectively.
Thus, the proposed mechanism is where KNI, KIU, and KNU are the equilibrium constants for the N ‘ I, I ‘ U, and N ‘ U transitions, respectively. The observed fluorescence / activity at any concentration of the denaturant is given by the sum of the contributions from the three states as
Fobs(c) = FNfN(c) + FIfI(c) + FUfU(c) (1)
where fN(c), fI(c), and fU(c) are the fractions of the three states at a denaturant concentration of c (fN + fI + fU = 1), and FN, FI, and FU are the fluorescence values of the pure N, I, and U states, respectively. The fN, fI, and fU are related to the equilibrium constants, KNI and KNU, of the unfolding transitions from N ‘ I and N ‘ U, respectively, and hence are related to the corresponding free energy changes, ��GNI and ��GNU, as follows:
fN = 1/(1 + KNI + KNU) = 1/[1 + exp(-‘GNI/RT)+ exp(-‘GNU/RT)]
fI = KNI/(1 + KNI + KNU) ) =exp(-‘GNI/RT)/[1 + exp(-‘GNI/RT) +exp(-‘GNU/RT)]
fU = KNU/(1 + KNI + KNU) ) =exp(-‘GNU/RT)/[1 + exp(-‘GNI/RT) +exp(-‘GNU/RT)] (2)
where R and T are the gas constant and the absolute temperature, respectively. For many globular proteins, the free energy changes of unfolding are known to vary approximately linearly with c (22), such that
��GNI = ��GNIH2O – mNI c
��GNU = ��GNUH2O – mNU c (3)
where ��GNIH2O, and ��GNU H2O are the ��GNI and ��GNU at 0 M GdnHCl, respectively, and mNI and mNU represent the dependence of the respective free energy changes on c and thus cooperativity indexes of the transitions.
Calculation of thermodynamic parameters
The equilibrium unfolding curve monitored by tryptophan fluorescence was fitted into following
three state equations
f = (a1+b1*x+(c1+p1*x)*exp(-j*(h-x)/0.592)+(e1+g1*x)*exp(-i*(k-x)/0.592))/(1+exp(-j*(hx)/
0.592)+exp(-i*(k-x)/0.592))
Fit f to y
where,
f: observed spectroscopic signal
a1: intercept of native baseline.
b1: slope of native baseline.
c1: intercept of intermediate state baseline
p1: slope of intermediate state baseline
j: cooperativity index (mN-I) for N-I process (1.5:initial value)
h: Midpoint of denaturant concentration (Cm N-I) for N-I process (0.5:initial value)
e1: intercept of denatured state baseline
g1: slope of denatured state baseline
i: cooperativity index (mN-U) for N-U process (2.5:initial value)
k: Midpoint of denaturant concentration (Cm N-U) for N-U process (1: initial value)
Sambrook, J., and Russell, D.W. (2001). Molecular cloning: a laboratory manual. Volume 1’3 (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press).
Sambrook, J.F., and Maniatis, E. (1989). T. 1989, Molecular cloning: a laboratory manual. Cold Spring Laboratory Press, New York.
Shukla, N., Bhatt, A.N., Aliverti, A., Zanetti, G., and Bhakuni, V. (2005). Guanidinium chloride’and urea’induced unfolding of FprA, a mycobacterium NADPH’ferredoxin reductase. FEBS Journal 272, 2216-2224.
CHAPTER 4
RESULTS
4.1. Biochemical and biophysical characterization of Zebrafish DHFR
4.1.1. Plasmid Isolation
The competency of DH5′ E.coli cells was confirmed by successful transformation of zDHFR gene present in pET 43.1a vector. The E. coli cells were grown on LB- Amp media at 37 ��C. The presence of zDHFR gene was confirmed by double digestion using Xho 1 and Nde 1 as restriction enzymes (Kao et al., 2008) and confirmed by visualizing it in 1% agarose.
Figure.4.1. Transformation of zDHFR and its confirmation by 1% agarose gel electrophoresis. (a). Transformed colonies of DH5′ E.coli cells bearing zDHFR gene grown on LB- Amp media. (b). 1% agarose gel electrophoresis profile confirming the presence of zDHFR gene in transformed cells. Lane 1, 1 kb DNA ladder; lane 2-5, closed, nicked and linear DNA (top to bottom) of 570 bp zDHFR.
4.1.2. Over-expression of Zebrafish DHFR
The competent BL21 (DE3) Rosetta E. coli cells were successfully transformed with the zDHFR gene in pET43.1a vector using heat shock method. The transformed cells were induced with 0.1 mM IPTG and an intense band on 12% SDS-PAGE confirmed the presence of desired zDHFR recombinant protein. The protein bands were visualized in Gel Doc after staining with Coomassie brilliant blue.
Figure 4.2. Over-expression of zDHFR at 37��C in E.coli BL21-DE3 Rosetta cells. (a). Transformed colonies of BL21 (DE3) Rosetta E. coli cells bearing zDHFR gene. (b).12 % SDS-PAGE profile confirming the over-expression of zDHFR. Lane 1: medium range protein molecular mass marker (97 -14 kDa), lane 2: uninduced BL21-DE3 Rosetta transformed cells, lane 3: induced BL21-DE3 Rosetta transformed cells.
4.1.3. Optimization of over-expression of zDHFR
When BL21-DE3 transformed cells were induced with different concentrations of IPTG, it was found that over-expression of zDHFR is equally good at all the concentrations of IPTG tested (Fig’. ). For optimization of the time of induction of IPTG, the transformed cells were induced with 0.1 mM IPTG. Good zDHFR over-expression occurred within 4-12 h of induction (Fig. 4.3 b).
Figure 4.3. Optimization of conditions for over-expression of zDHFR. a) 12% SDS-PAGE gel showing zDHFR expression at different concentrations of IPTG. Lane 1, medium range protein molecular mass marker; lane 2, uninduced BL21 DE3 Rosetta transformed cells; lane 3, cells induced with 0.04 mM IPTG; lane 4, 0.06 mM IPTG; lane 5, 0.08 mM IPTG; lane 6, 0.1 mM IPTG; lane 7, 0.2 mM IPTG and lane 8, 0.3 mM IPTG. b) 12 % SDS-PAGE gel showing zDHFR expression after induction with IPTG (0.1 mM) for different time intervals. Lane 1, protein medium range marker; lane 2, cells induced with IPTG for 2 h; lane 3, 4 h; lane 4, 6 h; lane 5, 8 h; lane 6, uninduced cells; lane 7, cells induced with IPTG for 10 h; lane 8, 12 h.
4.1.4. Enhancement of zDHFR over-expression using various osmolytes
Once the over-expression was confirmed by IPTG induction, an attempt has been made to further enhance the over-expression in presence of various osmolytes. Transformed E.coli cells bearing zDHFR gene were grown in LB Amp medium at 37 ��C containing optimized concentrations of various osmolytes (Thapliyal and Chaudhuri, 2015). The cells were induced by 0.1 mM IPTG when reached an OD600 of 0.8- 1.0.and incubated at 25 ��C for 6 h. An expression control (without any osmolytes) was also grown under similar environmental conditions.
Figure 4.4. Enhancement of over-expression in zDHFR using osmolytes. (a). Expression of zDHFR in presence of various osmolytes confirmed by 12% SDS-PAGE. Lane1, low molecular weight protein marker; lane 2, uninduced BL21 (DE3) Rosetta E.coli cells; lane 3, 0.1 mM IPTG induced cells; lane 4, IPTG induced cells in the presence of 0.2% glucose; lane 5, 1 mM betain; lane 6, 0.5 M sorbitol; lane 7, 2 M glycerol; lane 8, 0.5 M sucrose; lane 9, 1 mM proline; lane 10, 100 mM glycine; lane 11, 0.5 M NaCl and lane 12, 200 mM urea. IPTG induced protein sample was considered as expression control. (b). Bar graph represents level of in vivo zDHFR expression in presence of various osmolytes.
4.1.5. Purification of Zebrafish DHFR
Recombinant, monomeric zebrafish DHFR was isolated and purified from BL21 (DE3) Rosetta strain of Escherichia coli, carrying desired plasmid, zDHFR-His/pET43.1a that encodes zDHFR gene preceded by six histidine codons, under Lac Z promoter. Single step affinity chromatography based purification was performed using Ni-NTA chelating column, HiTrap HP (New Jersy, USA) in AKTA FPLC purification system (Kao et al., 2008). His-tagged zDHFR was eluted at around 150mM imidazole using linear concentration gradient of 0-500 mM imidazole. Protein aliquots with more than 95% purity were pooled. Dialysis was performed using refolding buffer to minimize the concentration of imidazole and further concentrated using amicon tubes (Millipore, U.S.A.) with 10 kDa molecular weight cut off membrane. Purification of the protein was confirmed by 12% SDS-PAGE (Laemmli, 1970).
Figure 4.5. (a). FPLC chromatogram of zDHFR purification using affinity chromatography at 280 nm with imidazole gradient from 0-500 mM. X-axis represents volume (ml) while Y- axis displays UV absorbtion at 280 nm. (b). The fractions obtained after affinity chromatography were run on 12 % SDS- PAGE. Lane 1, Low molecular weight protein marker; lane 3, pellet obtained after sonication of lysed induced cells; lane 2 and 4, sonicated supernatant loaded on affinity column; lane 5, flow through during load; lane 6-10, different fractions obtained after affinity chromatography.
4.1.6. Characterization of zDHFR
4.1.6.1. Confirmation of Molecular mass by Mass Spectroscopy
The molecular mass of purified zDHFR protein was verified by MALDI TOF mass spectrometry (Bruker)(Figure 4.6). zDHFR has an extinction coefficient of 24,075 M-1 cm-1 at 280 nm as computed using the ProtParam tool of ExPASy (http://web.expasy.org/protparam/). It was used for the determination of concentration of purified protein.
Figure 4.6. Molecular mass of zDHFR. Molecular mass represented by a MALDI mass spectra showing zDHFR peak corresponding to molecular weight in Daltons.
4.1.6.2. Estimation of disulphide bond
1 ml protein solution (0.3%) in 0.1 M sodium phosphate buffer, pH 7.4 containing SDS and 1 mg EDTA was added 0.05 ml of 0.01 M DTNB solution in the same buffer under N2 atmosphere. Absorbance was observed after 30 minutes at 412 nm. Concentration was measured from known value of molar extinction coefficient and expressed as moles of sulfhydryl groups per mole of the protein. The reduction of DTNB gave rise to a spectroscopic signal that was proposed to represent the presence of one free thiol in zDHFR. As zDHFR contains 3- cysteine residues, the number of disulphide bonds was calculated to be, (3.0-1.0)/ 2 =1.0.
4.1.6.3. Enzyme activity
zDHFR activity was determined by spectroscopic method described earlier. The assay depends on the reversible NADPH dependent reduction of dihydrofolic acid to tetrahydrofolic acid in the presence of DHFR enzyme. This reaction was monitored by the decrease in absorbance of NADPH at 340 nm. The control experiment was carried out in the absence of DHFR protein to account for the blank contribution from NADPH. The value of blank was almost negligible as compared to the enzyme assay mixture in the presence of DHFR protein. The decrease in the concentration of NADPH in the presence of protein is thus attributed to DHFR catalysed reaction only. The assay was performed at 25 ��C. The standard assay mixture for zDHFR was composed of assay buffer, 20 mM Tris-HCl, pH 7.4, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG and 10 % glycerol along with substrate,100 ��M dihydrofolic acid and co-factor,140 ��M NADPH. One unit of enzyme has been described as the amount of enzyme required to oxidize 1 ��mole of dihydrofolate per min, which is based on its molar extinction coefficient of 12,300 M’1 cm’1 at 340 nm. All enzyme assays were performed in triplicate and to minimize the degradation of substrate and cofactor, NADPH and DHF were prepared fresh for each experiment, incubated in ice and consumed within 2 h of experimentation.
4.1.6.4. Intrinsic tryptophan fluorescence spectra
Steady state fluorescence spectra was recorded on a Perkin-Elmer life luminescence spectrometer LS 55 (Perkin-Elmer, USA) at 25��C using an optical cuvette of path length 1 cm. Intrinsic tryptophan fluorescence spectra of the purified zDHFR in 20 mM Tris pH 7.4, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG and 10% glycerol buffer was recorded after exciting the protein sample at 295 nm using excitation and emission slit widths of 5 nm and 10 nm, respectively. Emission spectra were monitored in the range of 310-400 nm. Baseline correction was performed with buffer in the absence of protein. Final zDHFR concentration was 5 ��M. The emission maxima for native zDHFR was 329 nm as shown clearly in the Figure 4.1.5.4.
Figure 4.7. Tryptophan fluorescence spectra of zDHFR. Tryptophan fluorescence spectra of 5 ��M native zDHFR in 20 mM TrisHCl pH 7.4, 25 mM KCl, 1mM GSH, 0.1 mM GSSG and 10% glycerol at 25��C. The zDHFR native protein has been excited at 295 nm and intrinsic tryptophan fluorescence emission has been recorded between 310-400 nm. Excitation and emission slit widths were 5 nm and 10 nm, respectively.
4.1.6.5. Extrinsic fluorescence spectra
For extrinsic fluorescence spectra using ANS as external fluorophore, protein solution was incubated with 50 ��M ANS at room temperature for 5 min after which the sample was excited at 370 nm and ANS emission spectra were recorded between 400-600 nm. Free ANS gives maxima at 468 nm. Here, excitation and emission slits used were 5 and 10 nm, respectively. Scan rate was 60nm/ min. Final DHFR concentration was 0.5 ��M. The emission maxima for ANS bound zDHFR was 468 nm as shown in Figure 4.1.5.5.
Figure 4.8. Extrinsic fluorescence spectra of zDHFR. The extrinsic fluorescence emission spectrum of 0.5 ��M native zDHFR in 20 mM TrisHCl pH 7.4, 25 mM KCl, 1mM GSH, 0.1 mM GSSG and 10% glycerol at 25��C obtained upon excitation of the chromophore at 470 nm and emission recorded between 400- 600 nm. Excitation and emission slit widths were 5 nm and 10 nm, respectively.
4.1.6.6. Far-UV CD spectra
Far-UV CD spectra of purified zDHFR protein were recorded on JASCO J-815 CD polarimeter (JASCO, Japan), flashed with nitrogen gas. The buffer used for the measurement was 20 mM Tris pH 7.4, 25 mM KCl. Protein concentration used was 5 ��M. The spectra were recorded between 200-250 nm in a 0.2 cm cuvette with a 1 mm band width and 1 s response time. Each spectrum represents an average of three accumulations. The background signal for all the spectra was corrected by buffer alone. The calculation for Molar residue ellipticity (MRE; deg cm2 dmol-1) was based on the following formula:
[��] = ��/n.c.l
where �� represents the measured ellipticity (millidegree), n represents the number of amino acid residues in the protein, the molar concentration of protein is depicted by c , and l represents the path length (mm). The values in millideg has been converted to molar residue ellipticity using a software associated with the instrument which utilizes the concentration of the protein solution and the path length of the cuvette. Molar residue ellipticity (MRE) of native zDHFR shows negative ellipticity around 218 nm which becomes positive after 200nm (Figure 4.1.5.6.). The percentages of the different secondary structures elements (��-helix- 20 %, ��-sheet- 39 %, ��- turn- 17 % and random coil- 24 %) were determined from the spectra (Yang et al., 1986).
Figure 4.9. Far-UV CD spectra of 5 ��M native zDHFR in 20 mM TrisHCl pH 7.4, 25 mM KCl, 1mM GSH, 0.1 mM GSSG and 10% glycerol at 25��C. Scan rate of 60 nm/min and band width of 1 mm was used. The spectrum is the average of three accumulations. Molar residue ellipticity (MRE) was calculated from the mdeg values, by the software provided with the instrument. Contribution from the buffer alone was subtracted.
4.1.7. In vivo folding of DHFR
In order to monitor the in vivo folding of DHFR, fractionation experiments were carried out. The principle behind these experiments is that the folded protein is soluble and physiologically active, whereas the misfolded and aggregated proteins are insoluble and have ideally no functional activity. Thus, when the E.coli cells containing the over-expressed proteins are lysed and centrifuged, folded and soluble proteins come in the supernatant and misfolded or aggregated stuff appears in the pellet. BL21 E.coli cells containing the desired recombinant plasmids were over- expressed using IPTG and lysed by ultrasonication as described earlier. After centrifugation at high speed, supernatant and pellet were loaded on 12 % SDS- PAGE as shown in Figure 4.1.6. which shows good over-expression as compared to uninduced sample. However, around 50% of protein was expressed in soluble fraction while remaining was present in pellet.
Figure 4.10. In vivo folding of zDHFR as shown by 12 % SDS-PAGE. Lane 1, low molecular weight protein marker; lane 2, pellet obtained after centrifugation; lane 3, supernatant obtained after centrifugating the sonicated sample ; lane 4, uninduced E. coli cells.
4.1.8. In vitro unfolding studies of DHFR
Protein folding inside the cell is a highly complicated process and differs depending upon the complexity of the organism to which the protein belongs. For example, it is much more complicated in eukaryotic cells than prokaryotic ones. Because of this reason, there are less reports of in vivo protein folding study. Instead, it is much more convenient to study biophysical aspect of a purified protein in vitro. Few challenges has to be overcome, first one is to purify the protein under study from the host organism in sufficient amount to carry out in vitro studies. Second challenge is to optimize in vitro refolding conditions to achieve native protein. In this regard, protein has to be unfolded initially followed by refolding. Refolding conditions are highly crutial aspect for obtaining good yield of refolded protein.
4.1.8.1. Buffers and solutions
The native or the refolding buffer used in equilibrium experiments was 20 mM Tris pH 7.4, 25 mM KCl in case of E.coli and human DHFR while the buffer composition for zebrafish DHFR comprises of 20 mM Tris pH 7.4, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG and 10 % glycerol. The refolding buffer conditions were optimized to achieve complete unfolding reversibility and to avoid any hysteresis in the equilibrium unfolding transition curve. The unfolding buffer was the native buffer containing 3 M GdnHCl or 7 M urea. Urea solutions were always prepared fresh just before the experiments. The refractive indices of the stock solutions of GdnHCl and urea were determined using an Abbe 3L refractometer for measuring the concentration of the denaturant. The equation given by Pace, 1986 was used to calculate the GdnHCl concentration
C= 57.177 (��N) + 38.68 (��N)2 + 91.6 (��N)3 + ”’..
Where, ��N is the difference between refractive index of the native buffer and GdnHCl containing native buffer. All measurements were carried out at room temperature (25��C).
4.1.8.2. GdnHCl vs activity titration of zDHFR
To monitor how enzymatic activity of DHFR changes upon denaturation, 0.2 ��M native zDHFR in 20 mM Tris pH 7.4, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG, 10 % glycerol buffer containing different GdnHCl concentrations (0-3.5 M) for 2 h at 25�� C. Following this, the activity of each of the protein solution was recorded using the activity assay procedure of DHFR as described before. GdnHCl concentration was kept same in the activity assay buffer as was during unfolding to avoid any refolding during the activity assay. The protein shows complete loss of activity beyond 2 M concentration of GdnHCl (Figure 4.1.7.2.)
Figure 4.11. Equilibrium unfolding profile of zDHFR using GdnHCl monitored through loss of enzyme activity. 0.2 ��M native zDHFR was incubated in 20 mM Tris pH 7.4, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG, 10% glycerol buffer containing different GdnHCl concentrations (0-3 M) for 2 h at 25 ��C and enzyme activity was measured at 340 nm. Experiment was performed in triplicate and background corrections were done using blank samples without protein.
4.1.8.3. Monitoring equilibrium unfolding of zDHFR by intrinsic tryptophan fluorescence at increasing concentration of GdnHCl
The spectra obtained for GdnHCl induced unfolding of zDHFR monitored through change in intrinsic tryptophan fluorescence shows complete unfolding at 2M concentration of GdnHCl (Figure 4.1.7.3.). At very low concentration of GdnHCl (0.1 M-0.5 M), protein shows enhancement of fluorescence emission with no change in ��max. This change may be due to the fact that there was internal quenching of protein, and lower concentration of denaturant is dequencing the fluorophores. Beyond this concentration there was a gradual red shift in ��max with decreasing tryptophan emission intensity, which became almost constant after 2 M concentration of GdnHCl, after that there was no further shift in ��max.
Figure 4.12. Intrinsic tryptophan fluorescence of zDHFR on interaction with various concentrations of GdnHCl. 2 ��M. zDHFR was pre-incubated for 2 h with GdnHCl at 25�� C in refolding bu’er (pH 7.4) containing the mentioned concentrations of denaturant. Fluorescence was monitored at an excitation wave length of 295 nm and emission was recorded in the range (310’400 nm) with a excitation and emission slit width 5 nm and 10 nm, respectively. A. Fluorescence spectra of zDHFR were measured at di’erent concentrations (0’3 M) of GdnHCl. For the sake of visibility, the spectra of only 0, 0.1, 0.2, 0.3, 0.5, 0.9, 1.2, 1.4, 1.6, 2.5 and 3 M concentrations of GdnHCl are shown. B. Transition curves of zDHFR as a function of GdnHCl based denaturation are shown as relative ‘uorescence intensity measured at 340 nm. The fluorescence intensity value of native zDHFR is considered as 1.
4.1.8.4. Monitoring equilibrium unfolding of zDHFR by intrinsic tryptophan fluorescence at increasing concentration of urea
The spectra obtained for urea induced unfolding of zDHFR monitored through change in intrinsic tryptophan fluorescence shows complete unfolding around 7M concentration of urea. During urea induced denaturation, from 0- 1 M concentration of urea, protein shows folded conformation like native protein. Beyond 1 M concentration of urea, protein starts its unfolding with a gradual shift in ��max. Thus, protein gets completely unfolded at 7 M concentration of urea. (Figure 4.1.7.4.).
Figure 4.13. Intrinsic tryptophan Fluorescence analysis of zDHFR on interaction with various concentrations of urea. The concentration of zDHFR was 2 ��M. zDHFR was incubated with urea for 3h at 25�� C in refolding bu’er (pH 7.4) containing various mentioned concentrations of denaturant. Fluorescence was monitored at an excitation wave length of 295 nm in the emission was recorded from 310’ 400 nm with an excitation and emission slit width 5 nm and 10nm, respectively. (a) Intrinsic tryptophan fluorescence spectra of zDHFR were measured in di’erent concentrations of urea (0’7 M). For better visibility of the spectra, only 0, 1, 2, 3, 4, 5, 6 and 7 M concentrations of urea are shown in the plot. (b) Urea denatured transition curves of zDHFR monitored at 340 nm considering the peak value of native zDHFR as 1.
4.1.8.5. Monitoring equilibrium unfolding of zDHFR by extrinsic ANS fluorescence using GdnHCl as denaturant.
ANS is a extensively used protein fluorescent probe, which gives characteristic fluorescence emission upon binding with hydrophobic clusters of the protein molecule that are hydrated and hence become accessible. Thus, monitoring the protein bound ANS fluorescence emission under varying unfolding conditions can provide understanding of characterization of protein binding sites. It gives close insight about the folding and unfolding pathways of a protein molecule. Here, in case of zDHFR, its affinity to ANS is maximum at 0.1 M concentration of GdnHCl as compared to native ANS bound protein. Unfolded zDHFR does not bind ANS as shown in spectra (Figure 4.1.1.2.(a)). ANS can also be used to analyze the extent of packing of hydrophobic cores in proteins that undergoes conformational changes. Denaturation at higher denaturant concentrations of GdnHCl results in a complete unfolding and hence the loss of ANS binding. The effect of GdnHCl on the ability of zDHFR to bind ANS monitored at 465 nm is shown in Figure 4.1.1.2.(b).
Figure 4.14. Extrinsic fluorescence study of ANS as a function of Gdn HCl during unfolding of zDHFR. (a). 0.5 ��M protein was denatured in the presence of Gdn HCl for 2 h at 25�� C. Unfolded protein was incubated with 50��M ANS for 5 mins at 25��C. Protein was excited at 370 nm and emission was recorded between 400 nm and 600 nm with excitation and emission slit width 5nm and 10 nm, respectively, scan rate was 60 nm/min. (b). The effect of GdnHCl on the ability of zDHFR to bind ANS monitored at 465 nm.
4.1.8.6. Monitoring equilibrium unfolding of zDHFR by extrinsic ANS fluorescence using urea as denaturant.
The quantum yield of zDHFR protein was enhanced when ANS was bound to the solvent exposed hydrophobic patches on the protein surface. Unfolded zDHFR does not bind ANS as shown in spectra (Figure 4.1.1.3.(a)). The presence of ANS can also be helpful in monitoring the extent of packing of hydrophobic cores in proteins which undergoes structural changes. The effect of denaturant on the ability of zDHFR to bind ANS is shown in Figure 4.1.1.3.(b). ANS binding with zDHFR in presence of urea shows enhanced extrinsic fluorescence at 0.7 M concentration of urea. Unfolding at higher denaturant concentrations of urea leads to complete loss of ANS binding.
Figure 4.15. Extrinsic fluorescence study of ANS as a function of urea during unfolding of zDHFR (a). 0.5 ��M protein was denatured in the presence of urea for 3 h. Unfolded protein was incubated with 50��M ANS for 5mins at 25��C. Protein was excited at 370nm and emission was recorded between 400nm and 600nm with excitation and emission slit width 5 and 10 respectively, scan rate was 60nm/min. (b). Extrinsic fluorescence study of ANS as a function of urea during unfolding of zDHFR monitored at 465 nm.
4.1.8.7. Monitoring equilibrium unfolding of zDHFR by Far UV-CD
Equilibrium unfolding of zDHFR was also monitored by far-UV CD at 222 nm, using an optical cuvette with 1 mm path length and the signal was averaged for 30 s. Relative millidegree values was calculated for each GdnHCl concentration as described earlier. Fraction folded for zDHFR monitored at 222 nm was plotted against GdnHCl concentration.
Figure 4.16. Fraction folded monitored by change in molar ellipticity of GdnHCl induced denaturation of zDHFR protein and recorded on JASCO J-815 CD polarimeter flashed with nitrogen gas. The buffer used for the measurement was 20 mM Tris, 25 mM KCl, 1 mM GSh, 0.1 mM GSSG, 10 % glycerol, pH 7.4 at 25 ��C. Protein concentration used was 2 ��M. The spectra were recorded between 200-250 nm in a 0.2 cm cuvette with a 1 mm band width. Each spectrum represents an average of three accumulations. The background signal for all the spectra was corrected by buffer alone.
4.1.8.8. Equilibrium unfolding experiments in presence of various denaturants
Conformational transitions and functional studies of zebrafish DHFR in presence of various denaturants monitored through loss of enzymatic activity, change in intrinsic and extrinsic fluorescence and far UV- CD are shown in fig. 3 (a-d), respectively. Unfolding conditions were optimized in presence of chemical denaturants like GdnHCl and urea to achieve maximum unfolding of the protein, as assessed by loss of enzymatic activity (Fig. 3a). A comparison of intrinsic tryptophan fluorescence spectra of GdnHCl, urea and acid induced unfolded zDHFR with the native protein shows complete unfolding at 3 M concentration of GdnHCl and 8 M concentration of urea, while in case of acid mediated denaturation, protein has not reached to the fully unfolded state (Fig. 3b). ANS based extrinsic fluorescence study shows that there was a clear blue shift in wavelength when protein was unfolded in presence of GdnHCl and urea while there is a red shift during acid denaturation at pH 2 (Fig. 3c). The enhanced extrinsic fluorescence intensity of zDHFR at pH 2 may represent a partially unfolded form of the protein having reasonably high value of exposed hydrophobic regions on the surface of the protein. Far UV-CD spectra revealed that the protein shows complete unfolding at 3 M Gdn HCl and 8M urea but shows partial unfolding and retains some secondary structure elements at pH 2 (Fig. 3d). At 3 M concentration of GdnHCl, the protein is completely unfolded and contains random coil conformation (Fig. 3d). The spectra of the native protein shows a minimum at 218 nm and become positive below 200 nm. The fractions of four structural components (‘-helix, ��-sheet, ��-turns and unordered) were estimated by the ‘self-consistent’ method (selcon3) and summarized in table 4.1.7.8. [23].
Table 4.1. Secondary structural components of zDHFR under different denaturing conditions
% ‘ Helix % �� sheet % �� turn % Random coil
Native zDHFR 20 39 17 24
7 M Urea 4 16 09 71
3 M GdnHCl 3 7 10 80
pH 2 8 25 15 52
Figure 4.17. Equilibrium unfolding of zDHFR in presence of different denaturants using various biophysical tools. (a). Histogram shows the influence of denaturing conditions on final refolding yield of zDHFR. Protein was denatured in presence of 3 M Gdn HCl, 8 M urea and acid, pH 2. Refolding was monitored by activity assay of the protein at 25��C, pH 7.4. % activity was calculated from recovery of enzymatic activity in 2 h as compared to native protein activity. Final concentration of zDHFR for enzymatic activity was kept 0.2��M. (b). Unfolding of 5 ��M zDHFR in presence of 3 M GdnHCl, 8 M urea and under extreme acidic condition (pH 2) monitored through intrinsic tryptophan fluorescence. The zDHFR protein was excited at 295 nm and emission was recorded at 310- 400 nm. (c). Extent of surface hydrobhobicity in presence of various denaturing conditions like 3 M GdnHCl, 8 M urea and under acidic condition (pH 2) and (d). Characterisation of different unfolded state of 5 ��M zDHFR protein monitored by far UV-CD. The native protein has been represented in black line, and the unfolded state present in Gdn HCl (red), urea (blue) and acid, pH 2 (pink) varies from each other as proved from the shift in ��max. Duration of incubation for unfolding initiated by above mentioned denaturants was 2, 3 and 4 hours respectively. Buffer composition for unfolding and refolding was 20 mM tris, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG and 10% glycerol, pH 7.4. All spectra were corrected with the blank without protein.
4.1.9. In-vitro refolding of Zebrafish DHFR
4.1.9.1. Equilibrium refolding of the zDHFR protein
Equilibrium refolding of the zDHFR protein has been carried out using 200 ��M denatured protein which was diluted 100-fold into the refolding buffer composed of 20 mM Tris, 25 mM KCl, 1 mM GSH, 0.1 mM GSSG, 10 % glycerol, pH 7.4. so that the final protein concentration was 2 ��M and residual GdnHCl concentration remain below 0.1 M. The process of refolding was monitored through changes in the intrinsic tryptophan fluorescence measurement (Figure 4.1.8.1.).
Figure 4.18. Equilibrium unfolding/ refolding analysed using sigma plot monitored by change in intrinsic fluorescence (2 ��M) at 340 nm. For refolding, protein was diluted 1:100 fold in the same bu’er containing various concentrations of GdnHCl. Continuous lines shows theoretical plot obtained by three state fitting using Sigma plot (systat software). Equilibrium unfolding has been represented as blue line while that of refolding as red line which were overlapping each other perfectly. Each curve was normalized to values from 1 to 0 before performing the fitting. All the unfolding study was performed in refolding buffer and the spectra were corrected with the blank without protein.
4.1.9.2. Equilibrium unfolding of zDHFR follows a three state process
Equilibrium unfolding of zDHFR was performed as a function of Gdn HCl and monitored using two different probes i.e. intrinsic fluorescence and far UV-CD and it has been observed that the two unfolding transitions were not overlapping depicting the existence of three state (Figure 4.1.8.2.). This was confirmed by equilibrium unfolding/ refolding studies analyzed using Sigma Plot (Systat Software) and fitted to a three state equation which again describes a three-state transition where monomeric native zDHFR protein unfolds via an intermediate state to fully denatured state. Unfolding/ refolding was monitored by measuring change in intrinsic fluorescence of the protein (Figure 4.1.8.1.).
Figure 4.19. Effect of Gdn HCl on conformational transition during equilibrium unfolding of zDHFR induced by increasing concentration of Gdn HCl (0- 3 M) as monitored through intrinsic tryptophan fluorescence in black, and far UV- CD at 222 nm in red with protein concentration 2 ��M.
4.1.9.3. Presence of intermediate species during refolding of GdnHCl denatured zDHFR
The spectroscopic analysis of the intermediate state as compared to native and unfolded state populated at 0.7 M Gdn HCl concentration was monitored by intrinsic fluorescence spectroscopy. It is clearly shown in the fig. 8 that intrinsic tryptophan fluorescence of intermediate state increases with respect to native and GdnHCl denatured state and fluorescence maxima got shifted from 339 nm (N) ‘ 338 nm (I) ‘ 352 nm (U). The enhanced intrinsic fluorescence of intermediate state, with negligible shift in the emission ��max indicates that the tryptophans at this state are in a different atmosphere than the native state of the proteins, perhaps away from the internal quenchers, and a remarkable shift in ��max of emission at higher GdnHCl concentration (3 M) has been clearly reflected in the unfolded state of the protein (Figure 4.1.8.3.).
Figure 4.20. Intrinsic tryptophan fluorescent spectra of 2 ��M of native (black), intermediate (red), unfolded (blue) and refolded (pink) zDHFR obtained after unfolding the protein at 3 M concentration of GdnHCl with subsequent removal of denaturant by dilution method to observe refolding of the protein. Protein was excited at 295 nm and emission was recorded between 310-400 nm. Excitation and emission slit width was 5 nm and 10 nm, respectively.
4.1.10. Thermodynamic parameters for the equilibrium unfolding process of zDHFR
Thermodynamic parameters for the equilibrium unfolding process of zDHFR has been calculated using the equation 2 mentioned in the materials and methods section. The value of ‘GNUH’O for the zDHFR protein has been obtained as 2.96 �� 0.5 Kcal/mol, which is closer to that of the human variant of the protein, however substantially lower than the E.coli protein (Table 4.1.9.).
Table 4.2. Thermodynamic parameters of zDHFR calculated from equilibrium unfolding transition
‘GNUH’O Kcal/mol mNU
(Kcal/mol) Cm NU
M ‘GNIH’O
Kcal/mol mNI
Kcal/mol Cm NI
M ‘GIUH2O
Kcal/mol mIU
2.96�� 0.5 10.6769��2.43 0.2775��0.022 1.139��0.04 8.5039��1.6 0.1340��0.024 1.8��0.46 2.17
4.1.11. Fractional population of equilibrium unfolding intermediate of zDHFR
GdnHCl concentration dependence on various fractions of native, intermediate and unfolded species were calculated based on equation 2. It has been demonstrated in figure 4.1.10. that highest population of intermediate species were present around 0.7 M concentration of GdnHCl.
Figure 4.21. Chemical denaturant concentration dependence of the fractions of the native (black), intermediate (red) and unfolded state (blue) of Gdn HCl induced unfolding monitored by intrinsic fluorescence. Fraction population was calculated based on equation 2 mentioned in thermodynamic calculations (experimental procedures).
Kao, T.-T., Wang, K.-C., Chang, W.-N., Lin, C.-Y., Chen, B.-H., Wu, H.-L., Shi, G.-Y., Tsai, J.-N., and Fu, T.-F. (2008). Characterization and comparative studies of zebrafish and human recombinant dihydrofolate reductases’inhibition by folic acid and polyphenols. Drug Metabolism and Disposition 36, 508-516.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. nature 227, 680-685.
Thapliyal, C., and Chaudhuri, P. (2015). EFFECT OF VARIOUS OSMOLYTES ON THE EXPRESSION AND FUNCTIONALITY OF ZEBRAFISH DIHYDROFOLATE REDUCTASE: AN IN VIVO STUDY. Journal of Proteins & Proteomics 6, 211-218.
4.1.1. Thermodynamic calculations
Thermodynamic parameters:
Biophysical Tool ‘GNUH’O Kcal/mol mNU
(Kcal/mol) Cm NU
M ‘GNIH’O
Kcal/mol mNI
Kcal/mol Cm NI
M ‘GIUH2O
Kcal/mol mIU
Trp Flu (340nm) using Gdn HCl 2.96��
0.5 10.6769��
2.43 0.2775��
0.22 1.139��
0.04 8.5039��
1.6 0.1340��
0.024 1.8��
0.46 2.17
Enz Act (340nm) using Gdn HCl 2.9255�� 0.9 10.2904��
4.5 0.2843�� 0.2 0.676�� 0.17 5.6711��
3.6 0.1193��
0.046 2.25��
0.73 4.6193��
0.8
4.1. Comparative equilibrium unfolding study using osmolytes between E. coli and Human DHFR
4.2.1. Over-expression of Recombinant DHFR proteins
Over-expression of recombinant DHFR protein was confirmed by 12% SDS-PAGE. The transformed cells were induced by 0.1 mM IPTG and compared by uninduced cells as shown in Figure 4.1.1. (a) and (b) for E.coli and human DHFR respectively.
Fig. 4.22. (a). Over-expression of EcDHFR using 12% SDS Gel Electrophoresis: Lane1, sample from uninduced cells; lane 2-6, samples from induced cells; lane 7, low MW protein marker. (b). Over expression of hDHFR using 12% SDS Gel Electrophoresis: lane 1, 3- samples from induced cells; lane 2, 5- samples from uninduced cells; lane 6, low MW protein marker; lane 4, left blank.
4.2.2. Purification of recombinant DHFR proteins
Purified fractions of DHFR proteins as obtained by Ni2+ NTA chromatography and shown by 12% SDS-PAGE confirmed that the size of E.coli DHFR is ~ 24 kDa (Figure 4.1.2.(a)), while that of Human DHFR is ~ 23 kDa (Figure 4.1.2.(b)).
Figure 4.23. 12% SDS-PAGE shows purification of DHFR proteins by Nickel Affinity Chromatography. (a). Purification profile of E.coli DHFR: lane 1, Medium range molecular weight protein marker; lane 2, sample from uninduced BL21 DE3 cells; lane 3, Pellet obtained after sonication of lysed induced cells; lane 4, Crude supernatant loaded on affinity column; lane 5, flow through during load; lane 6, wash; lane 7, Purified DHFR; lane 8, Purified DHFR after removal of imidazole through dialysis. (b). Purification profile of Human DHFR: Lane 1, Low molecular weight protein marker; lane 2, pellet obtained after sonication of lysed induced cells; lane 3, sonicated supernatant loaded on affinity column; lane 4, flow through during load; lane 5-9, different fractions obtained after affinity chromatography.
4.2.3. Equilibrium unfolding of DHFR proteins monitored through the changes in enzymatic activity
Equilibrium unfolding of DHFR was carried out with assaying the DHFR activity for the GdnHCl-denatured enzyme, and the percentage of residual activity of the enzyme relative to the activity of native enzyme was plotted against the GdnHCl concentration as shown in Figure 4.2.3. E. coli DHFR has been denatured completely and lost almost all its activity at 2 M of GdnHCl concentration (4.2.3.a) while Human DHFR enzyme loses its activity at around 1 M of GdnHCl concentration (4.2.3.b). This demonstrates, the higher stability of E.coli DHFR protein as compared to its human counterpart.
a. E. coli DHFR b. Human DHFR
Figure 4.24. Equilibrium unfolding of (a) E.coli DHFR and (b) hDHFR (conc. 0.2��M) for increasing Gdn HCl (0-5M) monitored by loss in enzyme activity at 340nm. Background corrections were performed by using buffers without protein for individual sample.
4.2.4. Equilibrium Unfolding of DHFR proteins monitored through changes in Intrinsic Tryptophan Fluorescence Spectroscopy
Tryptophan fluorescence emission of two variants of DHFR were measured in the wide concentration range of GdnHCl ( 0-5M) at 25��C, and the relative emission intensity values were plotted against the concentration of GdnHCl. The denaturation profile of E.coli DHFR and human DHFR using tryptophan fluorescence spectroscopy has been shown in Figure 4.2.4. It has been observed that the E.coli and human DHFR proteins exhibit maximum changes in the fluorescence emission in the presence of about 3 M (Figure 4.2.4.a) and 1 M (Figure 4.2.4.b) concentration of GdnHCl, respectively. This result indicates the lower conformational stability of human DHFR as compared to E.coli protein under the mentioned sets of condition.
a. E. coli DHFR b. Human DHFR
Figure. 4.25. Equilibrium unfolding of (a) E.coli DHFR and (b) hDHFR (conc. 1 ��M) for increasing Gdn HCl (0-5M) monitored by changes in intrinsic tryptophan fluorescence. The DHFR proteins were excited at 295 nm and emission was recorded between 310 ‘ 400 nm. Excitation and emission slit width were 5 nm and 10 nm, respectively. Contribution from buffer without protein was subtracted for each sample.
4.2.5. Equilibrium unfolding of DHFR proteins in presence of osmolytes monitored through change of enzymatic activity
Unfolding of E.coli DHFR and its human counterpart was also studied in the presence of various osmolytes, such as 30% glycerol, 1 M sucrose, 100 mM proline to understand their role in stabilization of these enzymes. This was investigated by performing the activity assay for the GdnHCl denatured enzyme in presence of the osmolytes and the percentage activity of GdnHCl denatured enzyme was calculated relative to the native protein activity. The enzyme activity data were plotted against the GdnHCl concentration. It was observed that the activity of the E.coli DHFR was lost around 2.2 M of GdnHCl concentration (Figure 4.2.5.a), and the loss of activity in presence of glycerol and sucrose took place at around 2.5 M concentration. While proline did not exhibit any significant stabilisation of the E.coli DHFR against GdnHCl induced denaturation. In the case of Human DHFR, the enzyme loses its activity at around 1.5 M of GdnHCl concentration (Figure 4.2.5.b). In the presence of osmolytes, glycerol and sucrose, the enzyme lost its activity at around 2M concentration. Hence the extent of osmolyte mediated stabilisation is significant in both the DHFR molecules.
Figure 4.26. Equilibrium unfolding profile of (a) E.coli and (b) human DHFR in presence of osmolytes monitored through change in enzymatic activity at 340nm. Square (‘), shows equilibrium unfolding of 0.2 ��M DHFR protein in presence of increasing concentration of GdnHCl (0-3M), red circle (‘), blue triangle (‘) and pink triangle (‘) shows equilibrium unfolding of DHFR proteins in presence of 100 mM proline, 1M sucrose, and 30% glycerol respectively, with increasing concentration of GdnHCl (0-3M).
4.2.6. Equilibrium unfolding of DHFR proteins in presence of osmolytes monitored by intrinsic tryptophan fluorescence spectroscopy
The influence of the osmolytes on the differential stabilization of E.coli and human DHFR was investigated by monitoring the changes in tryptophan fluorescence emission for the GdnHCl denatured enzyme in presence of the osmolytes. The values of intrinsic tryptophan fluorescence emission intensity were plotted against the GdnHCl concentration as shown in figure 6. It was observed that the maximum change in the values of tryptophan fluorescence intensity for the E.coli DHFR took place around 2.2 M of GdnHCl concentration (Figure 4.2.6.a), and the same phenomena happened in presence of glycerol and sucrose at around 2.5 M concentration. While presence of proline did not exhibit any significant difference in the fluorescence emission to the E.coli DHFR during GdnHCl induced denaturation. In case of Human DHFR (Figure 4.2.6.b), the maximum change in the values of tryptophan fluorescence took place at 1.5 M of GdnHCl concentration, while in presence of glycerol and sucrose, it appears around 2.0 M concentration. In the present case also proline did not exhibit any significant difference in the fluorescence emission to the human DHFR during GdnHCl induced denaturation. Hence the extent of osmolyte mediated stabilisation is significant in both the DHFR proteins.
Figure 4.27. Unfolding process of (a) E.coli and (b) human DHFR protein using change of tryptophan fluorescence as a tool. Square (‘), shows the change of tryptophan emission of DHFR during equilibrium unfolding (protein conc. 1 ��M) with increasing concentration of GdnHCl (0-3M), red circle (‘), blue triangle (‘) and pink triangle (‘) shows the changes in tryptophan fluorescence intensity during unfolding of protein in presence of 100 mM proline, 1M sucrose, and 30% glycerol respectively, with increasing concentration of GdnHCl (0-3M). All spectra were corrected against the respective buffers.
4.2.7. Equilibrium unfolding study of DHFR in presence of osmolytes monitored by extrinsic fluorescence spectroscopy
Differential stabilization of E.coli and human DHFR was investigated by extrinsic fluorescence spectroscopy for the GdnHCl denatured enzyme in presence of the osmolyte and the surface hydrophobic property of the denatured enzyme was compared with the native enzymes. Surface hydrophobicity values, based on the ANS binding with the proteins, were plotted against GdnHCl concentration (0-3M). It was observed that E.coli DHFR alone reached to the conformation of relatively higher surface hydrophobicity at 0.3 M of GdnHCl concentration (Figure 4.2.7.a). In the presence of 30% glycerol, or 1M sucrose, the higher surface hydrophobic conformation appeared around 1.0 M concentration of GdnHCl. However, in the presence of 100 mM proline the E.coli enzyme reached to the higher surface hydrophobic conformation at a GdnHCl concentration comparable to that in the absence of osmolytes. This is a clear indication that osmolytes may stabilise the native E.coli DHFR protein conformation and delaying the attainment of higher surface hydrophobic conformation during the GdnHCl-induced denaturation process.
The observation in the case of human DHFR is somewhat different from the E.coli counterpart. Human DHFR alone reached a relatively higher surface hydrophobicity at 0.1 M of GdnHCl concentration (Figure 4.2.7.b), in the presence of 30% glycerol, or 1M sucrose. The higher surface hydrophobic conformation appeared around 0.5 M concentration of GdnHCl. However, in the presence of 100 mM proline the E.coli enzyme reached to the higher surface hydrophobic conformation at a GdnHCl concentration comparable to that in the absence of osmolyte. This is a clear indication that osmolytes may stabilise the native protein conformation and delay the attainment of higher surface hydrophobic conformation during the GdnHCl-induced denaturation process. Hence the extent of osmolyte mediated stabilisation is significant in both the DHFR molecules, although the nature of stabilisation may be slightly different between them.
Figure 4.28. Unfolding process of (a) E.coli and (b) human DHFR protein using change of surface hydrophobicity as a tool in presence of osmolytes. Square (‘), shows the change of surface hydrophobic property of both variants of DHFR during equilibrium unfolding (protein conc. 1 ��M) with increasing concentration of GdnHCl (0-3M). Red circle (‘), blue triangle (‘) and pink triangle (‘) shows the changes of surface hydrophobicity during unfolding of DHFR in presence of 100 mM proline, 1M sucrose, and 30% glycerol respectively, with increasing concentration of GdnHCl (0-3M).
CHAPTER 5
DISCUSSION
5.1. Biochemical and biophysical characterization of Zebrafish DHFR
There have been many detailed studies on the folding of small monomeric proteins (ref…………………………………………..). Even there are many reports on DHFR protein of different origins (ref…………………………………………….). However, equilibrium unfolding and refolding study of zebrafish DHFR has not been reported till date. In the present work, we have established the equilibrium folding scheme of zDHFR after obtaining thermodynamic parameters of the protein. It undergoes a spontaneous folding process without any molecular participation and refolds completely when denatured in presence of GdnHCl and urea. In order to perform the thermodynamic studies of the folding of zDHFR, a reversible, path independent folding of ZDHFR had to be demonstrated.
Overexpression in zDHFR
In the present study, pET 43.1a vector bearing the zebrafish DHFR gene under the control of T7 promoter was used for over-expression of zDHFR in E. coli expression system. It has T7 RNA polymerase machinery under the control of Lac promoter. The overexpression of zDHFR protein was carried out by adding non-hydrolysable analogue of lactose, IPTG which typically acts as chemical inducer. The conditions for overexpression of zDHFR were optimized w.r.t. its concentration (Figure 4.3 a) and duration of incubation (Figure 4.3 b). The recombinant protein over expressed in the host cellular system should be sufficiently active at the time of induction. Thus, cell concentration before induction plays a crucial role for over-production of recombinant proteins (OD600 should be around 0.8- 1.0). A good level of expression in zDHFR protein was obtained in E. coli cells when induction was carried out in the mid-exponential phase. Over-expression was performed using uninduced sample as control.
Enhancement of over expression by osmolytes
Most of the over expressed recombinant proteins in bacterial cell cannot reach to a correct conformation and undergo proteolytic degradation or associate with each other and often tend to misfold and accumulate as soluble aggregates and/or inclusion bodies. Hence, there is an ever-growing interest in developing strategies to avoid protein aggregation or to enhance protein refolding yields. A strategy for improving the level of expression of recombinant proteins in a soluble native form is to increase the cellular concentration of osmolytes or chaperones. Osmolytes are naturally occurring organic compounds affecting osmosis and they protect organisms from stress induced by osmotic pressure. It represents diverse chemical categories including amino acids, methylamines, and polyols. Due to increased concentrations of osmolytes, organisms may undergo some conformational changes in cellular proteins. Osmolytes shift equilibrium toward natively-folded conformations by increasing the free energy of the unfolded state. Osmolytes mainly affect the protein backbone. This balance between osmolyte’backbone interactions and amino acid side chain’solvent interactions decides protein folding process.
Abnormal cell volume regulation significantly contributes to the pathophysiology of several disorders, and cells respond to these changes by importing, exporting, or synthesizing osmolytes to maintain volume homeostasis. In recent years, it has become quite evident that cells regulate many biological processes such as protein folding, protein disaggregation, and protein’protein interactions via accumulation of specific osmolytes. Many genetic diseases are attributed to the problems associated with protein misfolding/aggregation, and it has been shown that certain osmolytes can protect these proteins from misfolding. Thus, osmolytes can be utilized as therapeutic targets for such diseases (Naturally Occurring Organic Osmolytes: From Cell Physiology to
Disease Prevention’..Shagufta H. Khan1, Nihal Ahmad2, Faizan Ahmad3, and Raj Kumar1).
In the present study, bacterial cells were grown in the presence of high salt, sorbitol, glucose, sucrose, proline, urea, glycerol, glycine and betaine (Table 2.1). The understanding of the molecular mechanisms by which osmolytes and specific molecular chaperones act in stressed and non-stressed bacterial cells are very important to design the protocols to produce optimal amounts of natively folded recombinant proteins. The presence in the cell of physiological amounts of compatible osmolytes, such as proline, glycine, betaine and sorbitol can significantly increase the stability of native thermo-labile proteins.
Osmolytes induced enhancement of overexpression of zDHFR was authenticated by 12% SDS-PAGE, shown in figure 4.4.a. It was observed that protein shows good over expression in the presence of 0.1 mM IPTG (Figure 4.3). Amount of folded protein in a cell can be estimated based on the principle that the proteins with correctly folded structure are soluble in the cytoplasm and in aqueous buffer, however, denatured proteins are insoluble and occur as aggregates (Chaudhuri et al., 2001). The concept of folded or native protein in soluble fraction may be utilized to confirm the enhancement of expression by osmolytes because of the fact that osmolytes stabilizes the protein so that it may attain its native or folded conformation. Normalization of the cell culture was done such that the same number of cells was taken for the analysis of each sample along with the control (un-induced cells). In-vivo protein over-expression was verified in the presence of optimized concentration of osmolytes. IPTG induced protein sample without the presence of any osmolyte was considered as expression control. Protein shows enhanced expression of soluble protein in the presence of sorbitol, proline, urea and glycine but not much effect of glycerol and NaCl whereas glucose, sucrose and betain showed the inhibited expression (Chen et al., 2015),(Oganesyan et al., 2007). The level of in vivo zDHFR expression in the presence of various osmolytes has been presented by the bar graph (Figure 4.4 b) which shows level of in vivo zDHFR expression where an optimized concentration of different osmolytes was used along with 100 ��M IPTG.
From the bar graph (Figure 4.4 b), it is very clear that the impact of the glucose, sucrose and betain on the expression of recombinant protein is less than the traditional IPTG induced expression resulting in lower enzymatic activity, whereas sorbitol shows highest expression level as well as activity with zDHFR having six hydrogen bond donor and acceptor count. For the same reason glycerol also shows higher level of activity having three H-bond acceptor and donor count. This shows the protecting nature of osmolytes like glycerol and sorbitol, which increases the free energy of the unfolded form by interacting with the peptide bond in an unfavourable manner and hence, favouring the folded conformation of zDHFR. Proline and glycine being hydrophobic amino acids, which buried inside the protein core also have H-bond acceptor count which can show almost same activity level.
It is evident from the present study that osmolytes plays a crucial role in enhancement of expression to a substantial level in case of zDHFR. There are many reports suggesting the in- vivo role of osmolytes in correcting the folding defects of proteins for example, glycerol can correct the temperature sensitive folding defect of the human cystic fibrosis transmembrane conductance regulator mutant protein and tumor suppressor protein in cells (Sharma et. al., 2012).
6- histidine tagged zDHFR was purified by Ni2+ NTA affinity chromatography using FPLC and highly purified fractions were successfully eluted around 150 mM concentration of imidazole (Figure 4.5 a). The presence of purified fractions were validated by 12 % SDS- PAGE (Figure 4.5 b). The molecular mass of purified zDHFR protein was verified by MALDI TOF mass spectrometry,Bruker (Figure 4.6).
Establishment of the presence of disulphide bond
The number of disulphide bond present in zDHFR was determined to be one as found from Ellman’s assay.
Conformational studies of zDHFR
Conformational studies were performed using intrinsic fluorescence study, extrinsic fluorescence anf far UV- CD study. Tryptophan fluorescence spectrum of zDHFR (final concentration 5 ��M) was measured in the emission range of 310- 400 nm wavelengths at an excitation wavelength of 295 nm. Maximum fluorescence intensity was obtained at 329 nm (Figure. 4.7). Extrinsic fluorescence spectrum of zDHFR (final concentration 0.5 ��M) was measured to check surface hydrophobicity in the emission range of 400- 600 nm at an excitation wavelength of 370 nm. The spectra of native zDHFR using ANS as fluorophore, shows maxima at 468 nm (Figure. 4.8). Far UV- CD spectrum of zDHFR showed that the enzyme belonged to ‘+�� group of proteins, which was confirmed by analysing the CD spectra using ‘self-consistent’ method (selcon3). Analysis of secondary structure elements suggested that zDHFR contains substantial amount of both ‘-helix and ��-sheet secondary structures (Figure 4.9).
In vivo protein folding process on zDHFR
During in vivo fractionation experiment, a good level of expression of zDHFR protein was obtained in E. coli cells when induction was carried out under optimized set of conditions. Over-expression was performed using uninduced sample as control. Cells were lysed, centrifuged and loaded on 12 % SDS- PAGE to check the presence of folded protein in the soluble fraction. Around 50% of the overexpressed protein was present in soluble fraction while rest remains in the pellet (Figure 4.10). This indicates that half of the recombinant protein acquires its perfectly folded conformation while rest remain in the insoluble fraction in form of insoluble aggregates or inclusion bodies.
Significance of denaturing state on the refolding process of zDHFR
Protein conformational and stability studies, refolding studies are most commonly performed using chemical denaturants like Gdn HCl and urea. These denaturants may or may not have di’erent behaviour towards a protein.
Here, in case of enzymatic activity based equilibrium unfolding study using GdnHCl (Figure 4.11), the increase in activity of the enzyme in dilute denaturant was observed similar to intrinsic fluorescence study of zDHFR. This may be probably due to the change in polypeptide flexibility in the domain of active site. As the concentration of GdnHCl increases, the active site geometry of zDHFR got disrupted and enzyme got denatured and shows complete denaturation beyond 2M concentration of denaturant.
In case of tryptophan fluorescence based equilibrium unfolding of zDHFR, the decrease in intrinsic fluorescence at very low concentration of Gdn HCl (0-0.075 M) may be due to enhanced internal quenching of the protein (Figure 4.12 a). This decrease is not accompanied by a red shift in the ��max which indicates the possibility of internal quenching and the occurrence of more compact conformation of the protein in dilute denaturant. The transition to a higher intensity between 0.1-1 M concentrations of Gdn HCl, with insignificant red shift in ��max indicates the removal of internal quenching phenomenon. A gradual red shift in ��max of fluorescence emission, accompanied by decrease in fluorescence intensity of zDHFR was observed from 1- 1.8 M Gdn HCl concentration. Thereafter, emission maxima and fluorescence intensity were observed to be almost constant with a red shift of 13 nm (339 nm to 352 nm) upon complete Gdn HCl induced unfolding in the concentration range from 1.8 M- 3 M. This red shift is due to the replacement of tryptophan residues from the less polar interior of the protein to solvent exposed regions during unfolding process. Guanidine is an electrolyte with pKa of 11, below this pH value, it will be present in a fully protonated form as Gdn+. The presence of Gdn+ and Cl- in’uences the stability properties of proteins. The stabilizing e’ect of Gdn HCl and NaCl has been reported on RNase T where low concentration of GdnHCl leads to more compact form of native enzyme by binding to the negatively charged moieties of protein. There is stabilization of enzyme by a’nity binding of these cations at one or more sites. In the present case also, zDHFR may be stabilized by low concentration of cation binding to the negatively charged sites of the protein. Therefore at low concentration of GdnHCl, stabilization by Gdn+ cation binding to negatively charged sites in protein occurs and at higher concentrations it acts as a classical denaturant resulting in unfolding of protein chain.
ANS based unfolding studies of zDHFR clearly shows enhanced extrinsic fluorescence in presence of 0.1 M Gdn HCl which indicates the existence of molten globule like state. Protein, under the present study, in the molten globule state is functionally as active as in native state (Figure 4.14.).
As protein was gone through secondary structural changes under the influence of chemical denaturants like GdnHCl, the denatured state gradually loses its residual structure and while reaching to completely denatured state, its conformation converge towards the formation of random coil. (Fig. 3D). In the far UV region, native zDHFR revealed a well-resolved negative peaks at 218 nm. The enzyme loses all of its secondary structural integrity around 3 M GdnHCl or 8 M urea, as is evident by complete disappearance of all the characteristic peaks in far UV spectra (Fig. 3D).
Optimized unfolding conditions are mandatory for proper refolding of zDHFR
The refolding conditions were highly crucial for achieving good refolding yield of zDHFR. It has been optimized by using glutathione based redox system. We have attempted to optimize the refolding conditions of zDHFR so as to achieve complete reversibility of the unfolding transition of zDHFR(Fig……). One of the major findings of the refolding optimization process was that, the unfolded state of zDHFR influences the final refolding yield. In practice, when reduced form of glutathione was absent in the refolding buffer, almost 30% recovery of functional refolded zDHFR was achieved when denatured by GdnHCl. zDHFR contains 3 cysteine residues, out of which 2 cysteines makes a disulphide bond. Hence, absence of oxidised form of glutathione during refolding most likely would not allow the formation of correct disulphide bonding in the refolded zDHFR. Since, the refolding buffer contained redox system (i.e. GSH and GSSG) and glycerol; this implies that to achieve complete reversibility of the unfolding transition, it was important to minimize inter-molecular associations between zDHFR molecules during unfolding as well as refolding. The observation that 100% refolding yields were obtained from GdnHCl and urea denatured states of zDHFR (Fig. 4…….a), suggests that denatured states of proteins do decide how efficiently the protein refolds. We observed that without the involvement of this important component ‘glutathione’ in the right proportion, we were unable to achieve reversibility in case of GdnHCl-induced equilibrium unfolding study. The refolded enzyme obtained after serially diluting the chemical denaturant (GdnHCl), was found to be indistinguishable from native zDHFR, when monitored for biochemical and biophysical characteristics and the refolding yield was calculated based on intrinsic fluorescence. The refolding transitions determined by ‘uorescence measurements elucidate that GdnHCl based unfolding is reversible. The di’erence in stabilizing and destabilizing e’ect of GdnHCl is additive and results in the complex dependence of GdnHCl concentration and the stability of protein. Comprehending the conformational changes that result in a protein by various treatments would provide a powerful tool for understanding of cellular organization at molecular level. The above observations have thrown some light on the structural alterations and loss of function which can result due to exposure to denaturants and thus e’ect the normal functioning of the protein.
Three state process
The observation that far-UV CD and tryptophan fluorescence data do not coincide (Fig. 4.18) indicates non-two-state behaviour at equilibrium and hence to non-cooperativity within the system. In a fully cooperative system, since there are only fully folded or fully unfolded molecules present, and the formation of tertiary and secondary structure will be concerted, equilibrium denaturation experiments performed by fluorescence and CD will give the same result. However, if the cooperativity is lost, intermediates will accumulate and the two data sets will no longer be super-imposable. Hence, zDHFR equilibrium unfolding is characterized by the presence of at least one stable intermediate. Lower value of ��GNI, free energy change from N-I state, than ��GIU, free energy change from I-I- Ustate, shows that the intermediate is energetically closer to the native state rather than the fully unfolded state (Table 4.2). ‘m’ value is an important reaction coordinate that provides a measure of the change in the solvent accessible surface area upon unfolding and consequently average compactness of intermediates. Higher value of mNI than mIU shows that the change in solvent accessible surface area is more during N-I transition than I-U transition (Table 4.2). This indicates that the intermediate might be a ‘wet’ molten globule state. The observation that ANS fluorescence showed a steep increase at 0.1 M GdnHCl and then finally decreased to zero (Fig. 4.13) also favors strongly for existence of stable equilibrium intermediates.
Comparison of thermodynamic parameters of zDHFR
Thermodynamic parameters for zDHFR protein have been calculated upon fitting of the GdnHCl-mediated equilibrium unfolding data in three state equation. The ‘GNUH’O value for zDHFR was found to be 2.96 Kcal/mole as monitored by tryptophan fluorescence. A comparison has been made on the thermodynamic parameters of zDHFR with other DHFR varieties. It has been reflected from the mentioned comparison that the stability of zDHFR is closer to the human version, but the former protein reasonably less stable than the E.coli protein (Table 3).
Table 3. Comparision of thermodynamic parameters of different variants of DHFR.
‘GNUH’O Kcal/mol mNU (Kcal/mol) Cm (M) Reference
E.coli
(Mesophillic) 6.6��0.2 2��0.1 M 3.5 �� 0.1 [32]
Lactobacillus casei 4.6��0.6 2.4��0.1 1.8 �� 0.1 [32]
Human
2.4��0.3
1.7��0.1 1.4 �� 0.1
[32]
M. profunda (Piezophillic) 3.2 2.0 1.59 [24]
T. maritime (Thermophillic) 34.5 4.7 5.45 [24]
Halobacterium volcanii
(Halophillic) 4.9 [33]
Murine [34]
Mouse 4.4 �� 0.2 2.2��0.2 [35]
Zebra fish DHFR 2.96 �� 0.5 10.6769��2.43 0.2775��0.022 Present study
INTRODUCTION
A protein, differing in origin, may exhibits variable physicochemical behaviour, difference in sequence homology, fold and function. Studying structure-function correlationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence-structure-function correlationship. Hence, detailed understanding of structure- function relationships of wide variants of DHFR enzyme would be important for developing inhibitor or an antagonist against the enzyme involved in the cellular developmental processes. In the present study, we have reported the comparative structure-function relationship between E.coli and Human DHFR. The differences in the unfolding behaviour of these two proteins have been investigated to understand various properties of these two proteins like relative stability differences and variation in conformational changes under identical denaturation conditions. The equilibrium unfolding mechanism of DHFR proteins using GdnHCl as denaturant in the presence of various types of osmolytes has been monitored using loss in enzymatic activity, intrinsic tryptophan fluorescence and an extrinsic fluorophore ANS as probes.
5.2. Comparative equilibrium unfolding study using osmolytes between E. coli and Human DHFR
FROM biophysics PDF
The spectroscopy based monitoring of the unfolding of DHFR with varying concentrations of denaturant gives information about the change in secondary and tertiary structural elements upon denaturation. Osmolytes such as glycerol, sucrose, trehalose, proline etc. are naturally occurring compounds have been reported to be stabilisers for proteins (ref”””). By performing denaturation studies of E. coli and human DHFR proteins in the presence of various types of osmolytes experimental data may provide some information whether these proteins are also stabilised by the former, and also if there is any difference in nature and extent of stabilisation. These kinds of studies are useful for comparing the physicochemical properties of various protein molecules, or comparison among the variants of the same protein.
Higher stability of E. coli DHFR as compared to its human counterpart
The equilibrium unfolding studies of E. coli and human DHFR when monitored by the loss of enzymatic activity, it was observed that the protein completely lost its function at around 2M concentration of GdnHCl while human DHFR showed complete loss of activity at around 1M concentration of GdnHCl. The experimental data revalidates the structural information that the active site geometry between the human and E. coli DHFR are different (9). The result also indicates a higher stability of the E. coli protein than its human counterpart. In order to have finer information about the nature of their overall structure, the unfolding process monitored through a different probe, sensitive to the tertiary structure, could be useful. In fact, when the unfolding of both E. coli and human DHFR proteins were monitored through the intrinsic fluorescence spectroscopy, it was observed that the maximum change in fluorescence emission intensity occurred around 3M and 1M concentration of GdnHCl, respectively. This result indicates that for the E. coli protein, the active site disrupts earlier than the disruption of the overall tertiary structure. This is not the case for human protein in which the loss of activity and overall tertiary structure are happening simultaneously. It is suggesting subtle attributes relating to the overall difference in the architecture between the E. coli and human DHFR. GdnHCl is a charged chaotropic agent which denatures the protein through breaking of hydrogen bonds. Being charged in nature, it also stabilizes the denatured state through interacting with the surface of the protein. It gradually unfolds the protein based on the extent of weakening of the H bonds. Hence, the extent of denaturation depends on the concentration of the denaturant used for equilibration process. For the proteins there is a correlation between the extent of denaturation and GdnHCl concentration, however, the Cm values (mid-point of denaturant concentration) are unique characteristic for the protein related to thermodynamic stability of the protein. In order to compare the differential stability between the proteins, tryptophan fluorescence emission intensity gives some clues about the changes in the tertiary structure of the protein. In case of E. coli DHFR, it shows that there was not much change in intrinsic tryptophan fluorescence intensity of the protein up to 0.75 M concentration of GdnHCl. Beyond this concentration there is a sharp fall in intensity until it attains an almost constant value and also the ��max shifts to higher wavelengths. While in the case of human DHFR up to 0.5 M concentration of GdnHCl showed little change in intrinsic fluorescence intensity. Beyond this concentration the protein starts denaturation and it becomes nearly constant after 1M of GdnHCl. This suggests that the intrinsic thermodynamic stability of E. coli DHFR is higher than that of human DHFR protein.
Stabilization of DHFR proteins by osmolytes
Osmolytes, such as 1M sucrose, and 30% glycerol, provided enhanced stability to both the variants of DHFR. While assessing the osmolytes induced stability of both the DHFR proteins, it appears that the level of stabilisation is somehow related with its intrinsic stability. For example, sucrose and glycerol both shifted the point of complete deactivation for E. coli DHFR from 2.2 to 2.5 M GdnHCl concentration, whereas, the same osmolytes shifted the point of complete deactivation for hDHFR from 1.5 to 2 M GdnHCl concentration. While comparing the extrinsic fluorescence properties of E. coli and hDHFR in the presence various concentrations of GdnHCl, it was observed that the two proteins exhibited maximum surface hydrophobicity at different concentrations of the denaturant, such that E. coli DHFR, and hDHFR exhibited ANS binding maxima at 0.3 M and 0.1 M concentration of GdnHCl, respectively. The osmolytes, 30% glycerol and 1M sucrose, shifted the maximum surface hydrophobicity values to 1M and 0.5 M concentration of GdnHCl for E. coli and human DHFR, respectively. ANS is an external fluorescent probe which is sensitive to the surface hydrophobic character of protein molecules (19). ANS or bis ANS binds to the exposed hydrophobic patches on the protein surface and exhibit fluorescence emission at around 500 nm. Conformational transition as a function of denaturant concentration can be monitored with respect to the extent of ANS binding to the protein molecule. For example, if the partial denaturation of a protein exposes hydrophobic patches, then there would be an enhancement of the ANS binding upon equilibration with intermediate level of denaturation. Furthermore, 1, 8-ANS and bis-ANS have proven to be sensitive probes for partially folded intermediates in protein folding pathways. These applications take advantage of the strong fluorescence enhancement exhibited by these amphiphilic dyes when their exposure to water is lowered. Consequently, fluorescence of ANS increases substantially when proteins to which it is bound undergo transitions from unfolded to fully or partially folded states that provide shielding from water. Molten globule intermediates are characterized by particularly high ANS fluorescence intensities due to the exposure of hydrophobic core regions that are inaccessible to the dye in the native structure (20). Thus differential ANS binding results for E. coli and human DHFR is the revalidation of the overall difference in conformational properties between the two variants of proteins, as evident from the crystallographic studies (9).
CHAPTER 6
CONCLUSION
1. Successful overexpression of all the three variants of DHFR was achieved using IPTG. The optimal IPTG concentration for the recombinant expression system was found to be 0.1 mM with incubation time of 6 h.
2. Highly purified fractions of DHFR recombinant proteins were obtained by using a single step purification process (Ni2+ NTA affinity chromatography). Purified fraction of zDHFR has been eluted at 150 mM concentration of imidazole.
3. In vivo study for expression enhancement of zDHFR concluded that the recombinant protein expression has been enhanced significantly in the presence of optimized concentrations of osmolytes like sorbitol, glycerol, glycine and proline.
4. Equilibrium unfolding study of E. coli and human DHFR denatured by GdnHCl as monitored by loss of enzymatic activity and changes in tryptophan fluorescence depicts the higher stability of E. coli DHFR than its human counterpart.
5. Chemical chaperone (osmolytes) based equilibrium unfolding study of E. coli and human DHFR has been proved to enhance the conformational stability of the recombinant proteins using 1 M sucrose and 30 % glycerol.
6. Conformational studies of purified zDHFR by tryptophan fluorescence showed that the tryptophans in zDHFR are significantly buried in the non-polar environment as GdnHCl denaturation of zDHFR resulted in a significant shift of its ��max emission from 339 nm for the native protein to 352 nm for the fully unfolded protein. The maximum change in the tryptophan fluorescence of zDHFR upon unfolding occurred at 3 M GdnHCl after which there was no change upon further increase in GdnHCl concentration.
7. It has been concluded that zDHFR shows complete unfolding beyond 2 M concentration of GdnHCl as monitored by loss of enzymatic activity.
8. Equilibrium unfolding study of zDHFR in presence of various denaturants showed complete unfolding in the presence of 3 M GdnHCl and 7 M urea while acid denaturation couldn’t achieve fully unfolded conformation of the recombinant protein as monitored by intrinsic fluorescence, extrinsic fluorescence and far UV-CD spectroscopy.
9. zDHFR forms molten globule at 0.1 M GdnHCl concentration at which the protein was functionally active with intact secondary structure elements as concluded by the extrinsic fluorescence spectroscopic study. GdnHCl unfolded zDHFR does not bind to ANS.
10. zDHFR affinity to ANS is maximum at 0.7 M concentration of urea as compared to native ANS bound protein. It forms molten globule at this concentration of urea which shows native like conformation with intact secondary structure elements. Urea unfolded zDHFR does not bind to ANS.
11. It has been concluded from the non-coincidence of far UV-CD and tryptophan fluorescence data that the equilibrium unfolding of zDHFR is not a simple two- state process and apart from fully folded and fully unfolded states, another partially folded states are also present at equilibrium. Hence, unfolding transition of zDHFR has been proved to be a non-cooperative process.
12. GdnHCl induced unfolding/ refolding events of zDHFR were found to be reversible under the optimized set of conditions. During the optimization process, it was found that the unfolded state of zDHFR is important in determining its final refolding yield with GdnHCl and urea denatured state giving maximum refolding yields. Under conditions that minimize inter-molecular interactions of zDHFR, complete reversibility of the unfolding transition could be obtained.
13. GdnHCl induced equilibrium unfolding of zDHFR monitored by tryptophan fluorescence was concluded to be a three state process. Thermodynamic parameters were calculated from the three state fit of the transition, N’I’U. The equilibrium intermediate state was found to be closer to the native state as suggested by the free energy change and ‘m’ values.
14. Thermodynamic parameters for the equilibrium unfolding process of zDHFR has been calculated. The value of ‘GNUH’O for the zDHFR protein has been obtained as 2.96 �� 0.5 Kcal/mol.
15. It has been concluded by the spectroscopic analysis monitored by intrinsic fluorescence spectroscopy that the intermediate state of zDHFR unfolding as compared to native and unfolded state is populated at 0.7 M Gdn HCl concentration.
16. A comparison of the thermodynamic parameters of various variants of DHFR concluded that conformational stability of zDHFR is almost similar to its human counterpart, but it is reasonably less stable than the E. coli protein. Hence this unexplored variant of DHFR can be a good alternative model system for biochemical and biophysical studies of DHFR protein.