Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution

It has been known for several years that 1H NMR spectra of the enzyme staphylococcal nuclease contain resonances due to conformational heterogeneity [Markley, J. L., Williams, M. N., & Jardetzky, O. (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 645-651]. One source of conformational heterogeneity has been attributed recently to cis/trans isomeriation of the Lys116-Pro117 peptide bond [Evans, P. A., Dobson, C. M., Kautz, R. A., Hatfull, G., & Fox, R. O. (1987) Nature (London) 329, 266-268]. In this paper we present evidence for three interconverting folded forms of nuclease. Forms N and N' are monomeric; form N" appears at higher nuclease concentrations and probably corresponds to dimerized enzyme. Saturation transfer was used to demonstrate that exchange occurs between the denatured state and N". The effects of temperature, pH, and Ca2+ and nucleotide binding on NMR spectra of nuclease were examined. When the temperature is increased or the pH is lowered, form N' is favored relative to N. Binding of a competitive inhibitor (thymidine 3',5'-bisphosphate plus calcium ion) strongly favors one form of nuclease. 1H NMR spectra of wild-type nuclease, the single-mutant nucleases L89F and H124L, and the double-mutant nuclease F76V+H124L were compared. In the unligated proteins, the equilibrium constant for the conformational equilibrium N in equilibrium with N' is approximately 0.1 in wild-type nuclease and nuclease H124L; by contrast, this equilibrium constant is about 0.7 in nuclease L89F and 1.2 in nuclease F76V+H124L under similar conditions.     

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.     

Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers.     

Energetic coupling between native-state prolyl isomerization and conformational protein folding

In folded proteins, prolyl peptide bonds are usually thought to be either trans or cis because only one of the isomers can be accommodated in the native folded protein. For the N-terminal domain of the gene-3 protein of the filamentous phage fd (N2 domain), Pro161 resides at the tip of a beta hairpin and was found to be cis in the crystal structure of this protein. Here we show that Pro161 exists in both the cis and the trans conformations in the folded form of the N2 domain. We investigated how conformational folding and prolyl isomerization are coupled in the unfolding and refolding of N2 domain. A combination of single-mixing and double-mixing unfolding and refolding experiments showed that, in unfolded N2 domain, 7% of the molecules contain a cis-Pro161 and 93% of the molecules contain a trans-Pro161. During refolding, the fraction of molecules with a cis-Pro161 increases to 85%. This implies that 10.3 kJ mol(-1) of the folding free energy was used to drive this 75-fold change in the Pro161 cis/trans equilibrium constant during folding. The stabilities of the forms with the cis and the trans isomers of Pro161 and their folding kinetics could be determined separately because their conformational folding is much faster than the prolyl isomerization reactions in the native and the unfolded proteins. The energetic coupling between conformational folding and Pro161 isomerization is already fully established in the transition state of folding, and the two isomeric forms are thus truly native forms. The folding kinetics are well described by a four-species box model, in which the N2 molecules with either isomer of Pro161 can fold to the native state and in which cis/trans isomerization occurs in both the unfolded and the folded proteins.     

Thermodynamic origin of cis/trans isomers of a proline-containing beta-turn model dipeptide in aqueous solution: a combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear Overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study

The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-turn model dipeptide [13C]-Ac-L-Pro-D-Ala-NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H-nmr, two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one-dimensional steady-state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random-coil peptides in H2O (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-AlaNH and AlaNH-NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C-CONH(CH3) fragment exhibits significant conformational flexibility; however, a low-energy conformer resembles closely the beta II-turn folded conformations of the x-ray structure of the related model peptide trans-BuCO-L-Pro-Me-D-Ala-NHMe. On the contrary, the cis isomer adopts open conformations. Steady-state intermolecular solute-solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H-nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -5.47 J.K-1.mole-1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence-specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes.     

Cis proline mutants of ribonuclease A. I. Thermal stability.

A chemically synthesized gene for ribonuclease A has been expressed in Escherichia coli using a T7 expression system (Studier, F.W., Rosenberg, A.H., Dunn, J.J., & Dubendorff, J.W., 1990, Methods Enzymol. 185, 60-89). The expressed protein, which contains an additional N-terminal methionine residue, has physical and catalytic properties close to those of bovine ribonuclease A. The expressed protein accumulates in inclusion bodies and has scrambled disulfide bonds; the native disulfide bonds are regenerated during purification. Site-directed mutations have been made at each of the two cis proline residues, 93 and 114, and a double mutant has been made. In contrast to results reported for replacement of trans proline residues, replacement of either cis proline is strongly destabilizing. Thermal unfolding experiments on four single mutants give delta Tm approximately equal to 10 degrees C and delta delta G0 (apparent) = 2-3 kcal/mol. The reason is that either the substituted amino acid goes in cis, and cis<==>trans isomerization after unfolding pulls the unfolding equilibrium toward the unfolded state, or else there is a conformational change, which by itself is destabilizing relative to the wild-type conformation, that allows the substituted amino acid to form a trans peptide bond.     

Native-like partially folded conformations and folding process revealed in the N-terminal large fragments of staphylococcal nuclease: a study by NMR spectroscopy

The N-terminal large fragments of staphylococcal nuclease (SNase), SNase110 (1-110 residues), SNase121 (1-121 residues), and SNase135 (1-135 residues), and the fragment mutants G88W110, G88W121, V66W110 and V66W121 were studied by heteronuclear multidimensional NMR spectroscopy. Ensembles of co-existent native-like partially folded and unfolded states were observed for fragments. The persistent native-like tertiary interaction drives fragments to be in partially folded states, which reveal native-like beta-barrel conformations. G88W and V66W mutations modulate the extent of inherent native-like tertiary interaction in fragment molecules, and in consequence, fragment mutants fold into native-like beta-subdomain conformations. In cooperation with the inherent tertiary interaction, 2 M TMAO (trimethylamine N-oxide) can promote the folding reaction of fragments through the changes of unfolding free energy, and a native-like beta-subdomain conformation is observed when the chain length contains 135 residues. Heterogeneous partially folded conformations of 1-121 and 1-135 fragments due to cis and trans X-prolyl bond of Lys116-Pro117 make a non-unique folding pathway of fragments. The folding reaction of fragments can be characterized as a hierarchical process.     

NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

A magnetization-transfer nuclear magnetic resonance study of the folding of staphylococcal nuclease

The equilibrium between alternative folded states of a globular protein, staphylococcal nuclease, has been investigated by using 1H NMR. Magnetization-transfer experiments have revealed the existence of a related structural heterogeneity of the unfolded state, and quantitative analysis of a series of these experiments has permitted the kinetics of folding and interconversion of the different states to be explored. A model based on cis/trans isomerism at the peptide bond preceding Pro-117 has been developed to account for the results. This model, recently supported by a protein-engineering experiment [Evans et al. (1987) Nature (London) 329, 266], has been used to interpret the kinetic data, providing insight into the nature of the folding processes. The predominance of the cis-proline form in the folded state is shown to derive from a large favorable enthalpy term resulting from more effective overall folding interactions. The kinetics of folding and isomerization are shown to occur on similar time scales, such that more than one pathway between two states may be significant. It has been possible, however, to compare the direct folding and unfolding rates within the cis- and trans-proline-containing populations, with results suggesting that the specific stabilization of the cis peptide bond is effective only at a late stage in the folding process.     

Conformational stability and mechanism of folding of ribonuclease T1

Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.     

Stabilization of a strained protein loop conformation through protein engineering.

Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different conformations of the residue 112-117 loop. The cis form is favored over the trans with an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, a trans conformation is stabilized relative to the cis conformation by the release of steric strain in the trans form. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild-type protein in its structure and in the dominance of the cis configuration. The results of these studies suggested that any nuclease variant with a non-glycine residue at position 116 should also favor the cis form because of steric requirements of the beta-carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the "beta-carbon" hypothesis by favoring the cis form. Furthermore, the crystal structure of K116E is nearly identical to that of the wild-type protein. Two additional variants, K116D and K116N, provide exceptions to this simple "beta-carbon" rule in that the trans conformation is stabilized relative to the cis configuration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailed trans conformation of the K116D variant appears to be similar to the trans conformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.     

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.     

The importance of anchorage in determining a strained protein loop conformation.

We examine the role of the conformational restriction imposed by constrained ends of a protein loop on the determination of a strained loop conformation. The Lys 116-Pro 117 peptide bond of staphylococcal nuclease A exists in equilibrium between the cis and trans isomers. The folded protein favors the strained cis isomer with an occupancy of 90%. This peptide bond is contained in a solvent-exposed, flexible loop of residues 112-117 whose ends are anchored by Val 111 and Asn 118. Asn 118 is constrained by 2 side-chain hydrogen bonds. We investigate the importance of this constraint by replacing Asn 118 with aspartate, alanine, and glycine. We found that removing 1 or more of the hydrogen bonds observed in Asn 118 stabilizes the trans configuration over the cis configuration. By protonating the Asp 118 side chain of N118D through decreased pH, the hydrogen bonding character of Asp 118 approached that of Asn 118 in nuclease A, and the cis configuration was stabilized relative to the trans configuration. These data suggest that the rigid anchoring of the loop end is important in establishing the strained cis conformation. The segment of residues 112-117 in nuclease A provides a promising model system for study of the basic principles that determine polypeptide conformations. Such studies could be useful in the rational design or redesign of protein molecules.     

Reversible unfolding and refolding behavior of a monomeric aldolase from Staphylococcus aureus.

Thermal and GdmCl-induced unfolding transitions of aldolase from Staphylococcus aureus are reversible under a variety of solvent conditions. Analysis of the transitions reveals that no partially folded intermediates can be detected under equilibrium conditions. The stability of the enzyme is very low with a delta G0 value of -9 +/- 2 kJ/mol at 20 degrees C. The kinetics of unfolding and refolding of aldolase are complex and comprise at least one fast and two slow reactions. This complexity arises from prolyl isomerization reactions in the unfolded chain, which are kinetically coupled to the actual folding reaction. Comparison with model calculations shows that at least two prolyl peptide bonds give rise to the observed slow folding reactions of aldolase and that all of the involved bonds are presumably in the trans conformation in the native state. The rate constant of the actual folding reaction is fast with a relaxation time of about 15 s at the midpoint of the folding transition at 15 degrees C. The data presented on the folding and stability of aldolase are comparable to the properties of much smaller proteins. This might be connected with the simple and highly repetitive tertiary structure pattern of the enzyme, which belongs to the group of alpha/beta barrel proteins.     

Enzymatic catalysis of prolyl isomerization in an unfolding protein.

Prolyl isomerases are able to accelerate slow steps in protein refolding that are limited in rate by cis/trans isomerizations of Xaa-Pro peptide bonds. We show here that prolyl isomerizations in the course of protein unfolding are also well catalyzed. To demonstrate catalysis we use cytoplasmic prolyl isomerase from Escherichia coli as the enzyme and reduced and carboxymethylated ribonuclease T1 as the substrate. This form of ribonuclease T1 without disulfide bonds is nativelike folded only in the presence of moderate concentrations of NaCl. Unfolding can be induced by reducing the NaCl concentration at ambient temperature and in the absence of denaturants. Under these conditions prolyl isomerase retains its activity and it catalyzes prolyl cis/trans isomerization in the unfolding protein. Under identical conditions within the NaCl-induced transition unfolding and refolding are catalyzed with equal efficiency. The stability of the protein and thus the final distribution of unfolded and folded molecules attained at equilibrium is unchanged in the presence of prolyl isomerase. These results demonstrate that prolyl isomerase functions in protein folding as an enzyme and catalyzes prolyl isomerization in either direction.     

A kinetic study of the folding of staphylococcal nuclease using size-exclusion chromatography.

The kinetics of the hydrodynamic volume change accompanying the reversible unfolding of staphylococcal nuclease have been observed by size-exclusion chromatography at 4 degrees C and pH 7.0 using the denaturant guanidine hydrochloride. The observed chromatographic profiles have been simulated by a six-component unfolding/refolding mechanism using a consistent set of equilibrium and kinetic parameters. The native protein is an equilibrium mixture of the cis and trans isomers of the peptide bond preceding proline-117. The native conformation containing the cis isomer dominates the equilibrium mixture, is more stable, and unfolds more slowly at its transition midpoint. The denatured protein is an equilibrium mixture of at least four components, the cis/trans isomers of proline-117 and one of the five remaining prolines. The dominant refolding pathway is initiated from the denatured component containing the trans isomer of proline-117. The six-component mechanism is consistent with tryptophan fluorescence kinetic measurements of the wild-type protein and with chromatographic measurements of a mutant P117G protein.     

The coordination of the isomerization of a conserved non-prolyl cis peptide bond with the rate-limiting steps in the folding of dihydrofolate reductase

The propensity for peptide bonds to adopt the trans configuration in native and unfolded proteins, and the relatively slow rates of cis-trans isomerization reactions, imply that the formation of cis peptide bonds in native conformations are likely to limit folding reactions. The role of the conserved cis Gly95-Gly96 peptide bond in dihydrofolate reductase (DHFR) from Escherichia coli was examined by replacing Gly95 with alanine. The introduction of a beta carbon at position 95 is expected to increase the propensity for the trans isomer and perturb the isomerization reaction required to reach the native conformation. Although G95A DHFR is 1.30 kcal mol(-1) less stable than the wild-type protein, it adopts a well-folded structure that can be chemically denatured in a cooperative fashion. The mutant protein also retains the complex refolding kinetic pattern attributed to a parallel-channel mechanism in wild-type DHFR. The spectroscopic response upon refolding monitored by Trp fluorescence and the absence of a Trp/Trp exciton coupling apparent in the far-UV CD spectrum of the wild-type protein, however, indicated that the tertiary structure of the folded state for G95A DHFR is altered. The addition of methotrexate (MTX), a tight-binding inhibitor, to folded G95A DHFR restored the exciton coupling and the fluorescence properties through five slow kinetic events whose relaxation times are independent of the ligand and the denaturant concentrations. The results were interpreted to mean that MTX-binding drives the formation of the cis isomer of the peptide bond between Ala95 and Gly96 in five compact and stable but not wild-type-like conformations that contain the trans isomer. Folding studies in the presence of MTX for both wild-type and G95A DHFR support the notion that the cis peptide bond between Gly95 and Gly96 in the wild-type protein forms during four parallel rate-limiting steps, which are primarily controlled by folding reactions, and lead directly to a set of native, or native-like, conformers. The isomerization of the cis peptide bond is not a source of the parallel channels that characterize the complex folding mechanism for DHFR.