Essay: 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.

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