A stable dimer in the pH-induced equilibrium unfolding of the homo-hexameric enzyme 4-oxalocrotonate tautomerase (4-OT)

4-Oxalocrotonate tautomerase (4-OT) is a multimeric, bacterial enzyme comprised of 6 identical 62-amino acid subunits, which associate under native conditions to form a homo-hexameric structure stabilized entirely by noncovalent interactions. We have previously shown that the GuHCl-induced equilibrium unfolding of 4-OT at pH 8.5 is well modeled as a two-state process involving only hexamer and unfolded monomer; and we have obtained spectroscopic evidence that intermediate state(s) is (are) populated in the equilibrium unfolding reaction at pHs 6.0 and 7.4 [Silinski, P., Allingham, M. J., and Fitzgerald, M. C. (2001) Biochemistry 40, 4493-4502]. Here, we report on the pH-induced equilibrium unfolding of 4-OT using size-exclusion chromatography (SEC), far-UV-circular dichroism (CD) spectroscopy, and catalytic activity measurements over the pH range from 1.5 to 10.1. Our results indicate that the native hexamer of 4-OT is the predominant species in solution at pHs > or =6.2, that a partially folded dimeric state of 4-OT is stabilized in solution at pH 4.8, and that the enzyme is largely denatured in strongly acidic solutions (pH < or =3.1). GuHCl-induced equilibrium unfolding studies on 4-OT at pH 4.8 indicate that the folded 4-OT dimer populated at this pH is stabilized by 11.7 kcal.mol(-1). The results of biophysical studies on a fluorescent analogue of the enzyme, 4-OT(F50Y), and the results of UV photo-cross-linking studies on a synthetically derived 4-OT analogue, 4-OT(P1Bpa), suggest the polypeptide chains in the 4-OT dimer are nativelike in structure with the exception of their C-termini.     

Detection and characterization of partially unfolded oligomers of the SH3 domain of alpha-spectrin

For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of alpha-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above approximately 0.5 mM the population of the intermediate state becomes significant at temperatures between 45 degrees C and 80 degrees C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.     

Conformational plasticity of cryptolepain: accumulation of partially unfolded states in denaturants induced equilibrium unfolding

pH and chemical denaturant dependent conformational changes of a serine protease cryptolepain from Cryptolepis buchanani are presented in this paper. Activity measurements, near UV, far UV CD, fluorescence emission spectroscopy, and ANS binding studies have been carried out to understand the folding mechanism of the protein in the presence of denaturants. pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. The preliminary biophysical study on cryptolepain shows that major elements of secondary structure are beta-sheets. Under neutral conditions the enzyme was stable in urea while GuHCl-induced equilibrium unfolding was cooperative. Cryptolepain shows little ANS binding even under neutral conditions due to more hydrophobicity of beta-sheets. Multiple intermediates were populated during the pH-induced unfolding of cryptolepain. Temperature-induced denaturation of cryptolepain in the molten globule like state is non-cooperative, contrary to the cooperativity seen with the native protein, suggesting the presence of two parts, possibly domains, in the molecular structure of cryptolepain, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of A state (molten globule state) of cryptolepain is unique, as lower concentration of denaturant, not only induces structure but also facilitate transition from one molten globule like state (MG(1)) into another (MG(2)). The increase of pH drives the protein into alkaline denatured state characterized by the absence of any ANS binding. GuHCl- and urea-induced unfolding transition curves at pH 12.0 were non-coincidental indicating the presence of an intermediate in the unfolding pathway.     

Apolipoprotein E4 forms a molten globule. A potential basis for its association with disease

The amino-terminal domain of apolipoprotein (apo) E4 is less susceptible to chemical and thermal denaturation than the apoE3 and apoE2 domains. We compared the urea denaturation curves of the 22-kDa amino-terminal domains of the apoE isoforms at pH 7.4 and 4.0. At pH 7.4, apoE3 and apoE4 reflected an apparent two-state denaturation. The midpoints of denaturation were 5.2 and 4.3 m urea, respectively. At pH 4.0, a pH value known to stabilize folding intermediates, apoE4 and apoE3 displayed the same order of denaturation but with distinct plateaus, suggesting the presence of a stable folding intermediate. In contrast, apoE2 proved the most stable and lacked the distinct plateau observed with the other two isoforms and could be fitted to a two-state unfolding model. Analysis of the curves with a three-state unfolding model (native, intermediate, and unfolded) showed that the apoE4 folding intermediate reached its maximal concentration ( approximately 90% of the mixture) at 3.75 m, whereas the apoE3 intermediate was maximal at 4.75 m ( approximately 80%). These results are consistent with apoE4 being more susceptible to unfolding than apoE3 and apoE2 and more prone to form a stable folding intermediate. The structure of the apoE4 folding intermediate at pH 4.0 in 3.75 m urea was characterized using pepsin proteolysis, Fourier transform infrared spectroscopy, and dynamic light scattering. From these studies, we conclude that the apoE4 folding intermediate is a single molecule with the characteristics of a molten globule. We propose a model of the apoE4 molten globule in which the four-helix bundle of the amino-terminal domain is partially opened, generating a slightly elongated structure and exposing the hydrophobic core. Since molten globules have been implicated in both normal and abnormal physiological function, the differential abilities of the apoE isoforms to form a molten globule may contribute to the isoform-specific effects of apoE in disease.     

Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.     

Comparative analysis of two different amide-to-ester bond mutations in the beta-sheet of 4-oxalocrotonate tautomerase

Here we describe the total chemical synthesis and biophysical characterization of two backbone-modified, ester bond-containing analogues of the homohexameric enzyme 4-oxalocrotonate tautomerase (4OT). The amide-to-ester bond mutations in the two analogues in this study, (OI2)4OT and (OI7)4OT, were designed to effectively delete specific backbone-backbone hydrogen bonds in the beta-sheet region of the native 4OT hexamer. The (OI2)4OT and (OI7)4OT analogues each contained one ester bond per monomer that effectively deleted 12 backbone-backbone hydrogen bonds per hexamer. The structural properties of each analogue were characterized by size-exclusion chromatography (SEC), far-UV CD spectroscopy, and catalytic activity measurements, and they were found to be very similar to the structural properties of the wild-type enzyme. The results of equilibrium unfolding studies revealed that the (OI2)4OT and (OI7)4OT analogues were stabilized by 47.7 +/- 2.5 and 45.0 +/- 2.5 kcal/mol, respectively, under standard state conditions (1 M hexamer) as compared to a value of 69.6 +/- 3.3 kcal/mol for the wild-type control. Our results suggest that the two different, but structurally similar, backbone-backbone hydrogen bonds deleted in (OI2)4OT and (OI7)4OT make nearly equivalent contributions to the thermodynamic stability of the 4OT hexamer.     

Folding/Unfolding Kinetics of Apomyoglobin

The equilibrium and kinetic folding/unfolding of apomyoglobin (ApoMb) were studied at pH 6.2, 11 °C by recording tryptophan fluorescence. The equilibrium unfolding of ApoMb in the presence of urea was shown to involve accumulation of an intermediate state, which had a higher fluorescence intensity as compared with the native and unfolded states. The folding proceeded through two kinetic phases, a rapid transition from the unfolded to the intermediate state and a slow transition from the intermediate to the native state. The accumulation of the kinetic intermediate state was observed in a wide range of urea concentrations. The intermediate was detected even in the region corresponding to the unfolding limb of the chevron plot. Urea concentration dependence was obtained for the observed folding/unfolding rate. The shape of the dependence was compared with that of two-state proteins characterized by a direct transition from the unfolded to the native state.     

Acid and chemical induced conformational changes of ervatamin B. Presence of partially structured multiple intermediates

The structural and functional aspects of ervatamin B were studied in solution. Ervatamin B belongs to the alpha + beta class of proteins. The intrinsic fluorescence emission maximum of the enzyme was at 350 nm under neutral conditions, and at 355 nm under denaturing conditions. Between pH 1.0- 2.5 the enzyme exists in a partially unfolded state with minimum or no tertiary structure, and no proteolytic activity. At still lower pH, the enzyme regains substantial secondary structure, which is predominantly a beta-sheet conformation and shows a strong binding to 8-anilino-1- napthalene-sulfonic acid (ANS). In the presence of salt, the enzyme attains a similar state directly from the native state. Under neutral conditions, the enzyme was stable in urea, while the guanidine hydrochloride (GuHCl) induced equilibrium unfolding was cooperative. The GuHCl induced unfolding transition curves at pH 3.0 and 4.0 were non-coincidental, indicating the presence of intermediates in the unfolding pathway. This was substantiated by strong ANS binding that was observed at low concentrations of GuHCl at both pH 3.0 and 4.0. The urea induced transition curves at pH 3.0 were, however, coincidental, but non-cooperative. This indicates that the different structural units of the enzyme unfold in steps through intermediates. This observation is further supported by two emission maxima in ANS binding assay during urea denaturation. Hence, denaturant induced equilibrium unfolding pathway of ervatamin B, which differs from the acid induced unfolding pathway, is not a simple two-state transition but involves intermediates which probably accumulate at different stages of protein folding and hence adds a new dimension to the unfolding pathway of plant proteases of the papain superfamily.     

Native state EX2 and EX1 hydrogen exchange of Escherichia coli CspA, a small beta-sheet protein

Escherichia coli CspA is a small all-beta-sheet protein that folds fast (tau = 4 ms) via an apparent two-state mechanism. Our previous studies have shown that a large aromatic cluster on the surface of the protein participates in the rate-limiting step of folding and thus may be part of the folding nucleus of this protein. To obtain a more detailed picture of molecular events at the peptide backbone during unfolding and folding of CspA, we used native state hydrogen exchange and nuclear magnetic resonance spectroscopy (NMR). The experiments with native CspA were performed over a range of pH values from low pH, where exchange is governed by a rapid equilibrium before chemical exchange (EX2 exchange), to high pH, where exchange is dictated by the rate of unfolding (EX1 exchange). Rates of folding and unfolding were determined for 11 residues. The distribution of rates of folding within the structure of CspA suggests that hairpin turns, including one near the aromatic cluster, may nucleate the folding of CspA.     

Partially folded conformations of bovine liver glutamate dehydrogenase induced by mild acidic conditions

The acid-induced unfolding of bovine liver glutamate dehydrogenase (GDH) was studied using various spectroscopic methods such as far- and near-UV circular dichroism (CD), intrinsic and 1-anilino naphthalene-8-sulphonate (ANS) extrinsic fluorescence spectroscopy, light scattering and fluorescence quenching in 20 mM mixed buffer at various pHs. CD spectra show that at pH 3.5, GDH retains its secondary structure substantially, whereas its tertiary structure content is reduced considerably. Intrinsic fluorescence of GDH and ANS binding suggest that, at pH 3.5, the hydrophobic surface of enzyme is more exposed in comparison to the native form. Acrylamide quenching indicates more exposure of tryptophan residues of enzyme at pH 3.5 in comparison to pH 7.5. Another partially unfolded intermediate was detected at pH 5.0, which with its ANS binding capacity lies between the pH 3.5 intermediate and the native form of the enzyme. Gel filtration results revealed that the enzyme at pH 3.5 is dissociated into trimeric species whereas it exists as hexamer at pH 7.5 and 5.0. All the data taken together suggest the existence of two partially unfolded states of GDH at moderate acidic pHs which may be considered as molten and pre-molten globule-like states.     

Refolding of pyridoxine-5'-P oxidase assisted by GroEL

The unfolding of brain pyridoxine-5'-P oxidase by guanidinium chloride has been investigated at equilibrium. Circular dichroism, fluorescence spectroscopy and gel exclusion chromatography were used to monitor the unfolding process. The enzyme dissociates reversibly into monomers, but the fluorescence properties of the cofactor FMN are not restored upon dilution with potassium phosphate buffer (pH 7.4). Spontaneous refolding leads to 20% recovery of the catalytic activity. Addition of GroEL to the renaturing buffer accelerates the recovery of catalytic activity that approaches a level of 80% with respect to the native enzyme. The rate of recovery of catalytic activity assisted by GroEL parallels the rate of FMN fluorescence quenching, suggesting that structural rearrangements of the catalytic domain is the last step to take place in the refolding process.     

The stability, structural organization, and denaturation of pectate lyase C, a parallel beta-helix protein

Pectate lyase C (pelC) was the first protein in which the parallel beta-helix structure was recognized. The unique features of parallel beta-helix-containing proteins-a relatively simple topology and unusual interactions among side chains-make pelC an interesting protein to study with respect to protein folding. In this paper, we report studies of the unfolding equilibrium of pelC. PelC is unfolded reversibly by gdn-HCl at pH 7 and 5, as monitored by far- and near-UV CD and fluorescence. The coincidence of these spectroscopically detected transitions is consistent with a two-state transition at pH 7, but the three probes are not coincident at pH 5. No evidence was found for a loosely folded intermediate in the transition region at pH 5. At pH 7, the for unfolding is 12.2 kcal/mol, with the midpoint of the transition at 0.99 M gdn-HCl and m = 12.3 kcal/(mol.M). Thus, pelC is unusually stable and has an m value that is much larger than for typical globular proteins. Thermal denaturation of pelC has been studied by differential scanning calorimetry (DSC) and by CD. Although thermal denaturation is not reversible, valid thermodynamic data can be obtained for the unfolding transition. DeltaH(van't Hoff)/DeltaH(cal) is less than 1 for pHs between 5 and 8, with a maximum value of 0.91 at pH 7 decreasing to 0.85 at pH 8 and to 0.68 at pH 5. At all pHs studied, the excess heat capacity can be deconvoluted into two components corresponding to two-state transitions that are nearly coincident at pH 7, but deviate more at higher and lower pH. Thus, pelC appears to consist of two domains that interact strongly and unfold in a cooperative fashion at pH 7, but the cooperativity decreases at higher and lower pH. The crystal structure of pelC shows no obvious domain structure, however.     

The side chain of aspartic acid 69 dictates the folding mechanism of Bacillus subtilis HPr

Many small, single-domain proteins show equilibrium and kinetic folding mechanisms that appear to be adequately described as two state. The two-state model makes several predictions that can be tested experimentally. First, the conformational stability determined at or extrapolated to a set of reference conditions should be independent of the measurement method (thermal or solvent denaturation or hydrogen exchange). Second, model-independent measures of the cardinal thermodynamic parameters (T(m), DeltaH) as determined from direct calorimetric means should be identical to those determined from the two-state analysis of thermal unfolding data. Third, the ratio of the kinetic folding and unfolding rate constants should be equal to K(eq) determined from an equilibrium measurement under the same conditions. Here, we show that the wild-type HPr protein from Bacillus subtilis does not meet all of these criteria under our standard conditions. However, if we replace the side chain of Asp69, or add moderate concentrations of salt, we find excellent two-state behavior in both equilibrium and kinetic folding. Thus, for this protein and possibly others, very subtle changes in the primary structure or in the solution conditions can dramatically alter the relative stabilities of the native intermediate, and unfolded ensembles can cause an observable change in the nature of the folding mechanism.     

Kinetic traps in the folding/unfolding of procaspase-1 CARD domain

We have examined the folding and unfolding of the caspase recruitment domain of procaspase-1 (CP1-CARD), a member of the alpha-helical Greek key protein family. The equilibrium folding/unfolding of CP1-CARD is described by a two-state mechanism, and the results show CP1-CARD is marginally stable with a DeltaG(H2O) of 1.1 +/- 0.2 kcal/mole and an m-value of 0.65 +/- 0.06 kcal/mole/M (10 mM Tris-HCl at pH 8.0, 1 mM DTT, 25 degrees C). Consistent with the equilibrium folding data, CP1-CARD is a monomer in solution when examined by size exclusion chromatography. Single-mixing stopped-flow refolding and unfolding studies show that CP1-CARD folds and unfolds rapidly, with no detectable slow phases, and the reactions appear to reach equilibrium within 10 msec. However, double jump kinetic experiments demonstrate the presence of an unfolded-like intermediate during unfolding. The intermediate converts to the fully unfolded conformation with a half-time of 10 sec. Interrupted refolding studies demonstrate the presence of one or more nativelike intermediates during refolding, which convert to the native conformation with a half-time of about 60 sec. Overall, the data show that both unfolding and refolding processes are slow, and the pathways contain kinetically trapped species.     

Thermodynamics and kinetics of folding of common-type acylphosphatase: comparison to the highly homologous muscle isoenzyme

The thermodynamics and kinetics of folding of common-type acylphosphatase have been studied under a variety of experimental conditions and compared with those of the homologous muscle acylphosphatase. Intrinsic fluorescence and circular dichroism have been used as spectroscopic probes to follow the folding and unfolding reactions. Both proteins appear to fold via a two-state mechanism. Under all the conditions studied, common-type acylphosphatase possesses a lower conformational stability than the muscle form. Nevertheless, common-type acylphosphatase folds more rapidly, suggesting that the conformational stability and the folding rate are not correlated in contrast to recent observations for a number of other proteins. The unfolding rate of common-type acylphosphatase is much higher than that of the muscle enzyme, indicating that the differences in conformational stability between the two proteins are primarily determined by differences in the rate of unfolding. The equilibrium m value is markedly different for the two proteins in the pH range of maximum conformational stability (5. 0-7.5); above pH 8.0, the m value for common-type acylphosphatase decreases abruptly and becomes similar to that of the muscle enzyme. Moreover, at pH 9.2, the dependencies of the folding and unfolding rate constants of common-type acylphosphatase on denaturant concentration (mf and mu values, respectively) are notably reduced with respect to pH 5.5. The pH-induced decrease of the m value can be attributed to the deprotonation of three histidine residues that are present only in the common-type isoenzyme. This would decrease the positive net charge of the protein, leading to a greater compactness of the denatured state. The folding and unfolding rates of common-type acylphosphatase are not, however, significantly different at pH 5.5 and 9.2, indicating that this change in compactness of the denatured and transition states does not have a notable influence on the rate of protein folding.     

Investigation of folding/unfolding kinetics of apomyoglobin

Apomyoglobin kinetic and equilibrium unfolding and folding processes were studied at pH 6.2, 11 degrees C by stopped-flow tryptophan fluorescence. There are two distinct consecutive processes in apomyoglobin folding process, namely, the protein fast transition between the unfolded (U) and an intermediate (I) states (U <----> I) and slow transition between the intermediate and the native (N) states (I <----> N). Accumulation of the intermediate state was observed in the wide range of urea concentrations. The presence of the intermediate state was shown even beyond the middle transition on the unfolding limb. The dependence of observed folding/unfolding rates on urea concentration (chevron plot) was obtained. The shape of this dependence was compared with that of two-state proteins, folding from the U to N state.     

Kinetic, stereochemical, and structural effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase.

Three arginine residues (Arg-11, Arg-39, Arg-61) are found at the active site of 4-oxalocrotonate tautomerase in the X-ray structure of the affinity-labeled enzyme [Taylor, A. B., Czerwinski, R. M., Johnson, R. M., Jr., Whitman, C. P., and Hackert, M. L. (1998) Biochemistry 37, 14692-14700]. The catalytic roles of these arginines were examined by mutagenesis, kinetic, and heteronuclear NMR studies. With a 1,6-dicarboxylate substrate (2-hydroxymuconate), the R61A mutation showed no kinetic effects, while the R11A mutation decreased k(cat) 88-fold and increased K(m) 8.6-fold, suggesting both binding and catalytic roles for Arg-11. With a 1-monocarboxylate substrate (2-hydroxy-2,4-pentadienoate), no kinetic effects of the R11A mutation were found, indicating that Arg-11 interacts with the 6-carboxylate of the substrate. The stereoselectivity of the R11A-catalyzed protonation at C-5 of the dicarboxylate substrate decreased, while the stereoselectivity of protonation at C-3 of the monocarboxylate substrate increased in comparison with wild-type 4-OT, indicating the importance of Arg-11 in properly orienting the dicarboxylate substrate by interacting with the charged 6-carboxylate group. With 2-hydroxymuconate, the R39A and R39Q mutations decreased k(cat) by 125- and 389-fold and increased K(m) by 1.5- and 2.6-fold, respectively, suggesting a largely catalytic role for Arg-39. The activity of the R11A/R39A double mutant was at least 10(4)-fold lower than that of the wild-type enzyme, indicating approximate additivity of the effects of the two arginine mutants on k(cat). For both R11A and R39Q, 2D (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY-HSQC spectra showed chemical shift changes mainly near the mutated residues, indicating otherwise intact protein structures. The changes in the R39Q mutant were mainly in the beta-hairpin from residues 50 to 57 which covers the active site. HSQC titration of R11A with the substrate analogue cis, cis-muconate yielded a K(d) of 22 mM, 37-fold greater than the K(d) found with wild-type 4-OT (0.6 mM). With the R39Q mutant, cis, cis-muconate showed negative cooperativity in active site binding with two K(d) values, 3.5 and 29 mM. This observation together with the low K(m) of 2-hydroxymuconate (0.47 mM) suggests that only the tight binding sites function catalytically in the R39Q mutant. The (15)Nepsilon resonances of all six Arg residues of 4-OT were assigned, and the assignments of Arg-11, -39, and -61 were confirmed by mutagenesis. The binding of cis,cis-muconate to wild-type 4-OT upshifts Arg-11 Nepsilon (by 0.05 ppm) and downshifts Arg-39 Nepsilon (by 1.19 ppm), indicating differing electronic delocalizations in the guanidinium groups. A mechanism is proposed in which Arg-11 interacts with the 6-carboxylate of the substrate to facilitate both substrate binding and catalysis and Arg-39 interacts with the 1-carboxylate and the 2-keto group of the substrate to promote carbonyl polarization and catalysis, while Pro-1 transfers protons from C-3 to C-5. This mechanism, together with the effects of mutations of catalytic residues on k(cat), provides a quantitative explanation of the 10(7)-fold catalytic power of 4-OT. Despite its presence in the active site in the crystal structure of the affinity-labeled enzyme, Arg-61 does not play a significant role in either substrate binding or catalysis.     

Equilibrium and kinetic folding of an alpha-helical Greek key protein domain: caspase recruitment domain (CARD) of RICK

We have characterized the equilibrium and kinetic folding of a unique protein domain, caspase recruitment domain (CARD), of the RIP-like interacting CLARP kinase (RICK) (RICK-CARD), which adopts a alpha-helical Greek key fold. At equilibrium, the folding of RICK-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The protein is marginally stable, with a DeltaG(H)()2(O) of 3.0 +/- 0.15 kcal/mol and an m-value of 1.27 +/- 0.06 kcal mol(-1) M(-1) (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 degrees C). While the m-value is constant, the protein stability decreases in the presence of moderate salt concentrations (below 200 mM) and then increases at higher salt concentrations. The results suggest that electrostatic interactions are stabilizing in the native protein, and the favorable Coulombic interactions are reduced at low ionic strength. Above 200 mM salt, the results are consistent with Hofmeister effects. The unfolding pathway of RICK-CARD is complex and contains at least three non-native conformations. The refolding pathway of RICK-CARD also is complex, and the data suggest that the unfolded protein folds via two intermediate conformations prior to reaching the native state. Overall, the data suggest the presence of kinetically trapped, or misfolded, species that are on-pathway both in refolding and in unfolding.