Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.     

The Greek key protein apo-pseudoazurin folds through an obligate on-pathway intermediate

Folding of the 123 amino acid residue Greek key protein apo-pseudo azurin from Thiosphaera pantotropha has been examined using stopped-flow circular dichroism in 0.5 M Na2SO4 at pH 7.0 and 15 degrees C. The data show that the protein folds from the unfolded state with all eight proline residues in their native isomers (seven trans and one cis) to an intermediate within the dead-time of the stopped-flow mixing (50 ms). The urea dependence of the rates of folding and unfolding of the protein were also determined. The ratio of the folding rate to the unfolding rate (extrapolated into water) is several orders of magnitude too small to account for the equilibrium stability of the protein, consistent with the population of an intermediate. Despite this, the logarithm of the rate of folding versus denaturant concentration is linear. These data can be rationalised by the population of an intermediate under all refolding conditions. Accordingly, kinetic and equilibrium measurements were combined to fit the chevron plot to an on-pathway model (U <==> I <==> N). The fit shows that apo-pseudoazurin rapidly forms a compact species that is stabilised by 25 kJ/mol before folding to the native state at a rate of 2 s-1. Although the data can also be fitted to an off-pathway model (I <==> U <==> N), the resulting kinetic parameters indicate that the protein would have to fold to the native state at a rate of 86,000 s-1 (a time constant of only 12 microseconds). Similarly, models in which this intermediate is bypassed also lead to unreasonably fast refolding rates. Thus, the intermediate populated during the refolding of apo-pseudoazurin appears to be obligate and on the folding pathway. We suggest, based on this study and others, that some intermediates play a critical role in limiting the search to the native state.     

Replacement of proline with valine does not remove an apparent proline isomerization-dependent folding event in CRABP I

Site-directed mutagenesis has frequently been used to replace proline with other amino acids in order to determine if proline isomerization is responsible for a slow phase during refolding. Replacement of Pro 85 with alanine in cellular retinoic acid binding protein I (CRABP-I) abolished the slowest refolding phase, suggesting that this phase is due to proline isomerization in the unfolded state. To further test this assumption, we mutated Pro 85 to valine, which is the conservative replacement in the two most closely related proteins in the family (cellular retinoic acid binding protein II and cellular retinol binding protein I). The mutant protein was about 1 kcal/mole more stable than wild type. Retinoic acid bound equally well to wild type and P85V-CRABP I, confirming the functional integrity of this mutation. The refolding and unfolding kinetics of the wild-type and mutant proteins were characterized by stopped flow fluorescence and circular dichroism. The mutant P85V protein refolded with three kinetic transitions, the same number as wild-type protein. This result conflicts with the P85A mutant, which lost the slowest refolding rate. The P85V mutation also lacked a kinetic unfolding intermediate found for wild-type protein. These data suggest that proline isomerization may not be responsible for the slowest folding phase of CRABP I. As such, the loss of a slow refolding phase upon mutation of a proline residue may not be diagnostic for proline isomerization effects on protein folding.     

Proline isomerization during refolding of ribonuclease A is accelerated by the presence of folding intermediates

The trans----cis isomerization of Pro 93 was measured during refolding of bovine ribonuclease A. This isomerization is slow (tau = 500 s) under marginally stable folding conditions of 2.0 M GdmCl, pH 6, at 10 degrees C. However, it is strongly accelerated (tau = 100 s) in samples which, prior to isomerization, had been converted to a folding intermediate by a 15 s refolding pulse under strongly native conditions (0.8 M ammonium sulfate, 0 degree C). The results demonstrate that extensive folding is possible before Pro 93 isomerizes to its native cis state and that the presence of structural folding intermediates leads to a marked increase in the rate of subsequent proline isomerization.     

Detection of a hidden folding intermediate of the third domain of PDZ

The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ.     

Mechanism of the kinetically-controlled folding reaction of subtilisin

Like many secreted proteases, subtilisin is kinetically stable in the mature form but unable to fold without assistance from its prodomain. The existence of high kinetic barriers to folding challenges many widely accepted ideas, namely, the thermodynamic determination of native structure and the sufficiency of thermodynamic stability to determine a pathway. The purpose of this article is to elucidate the physical nature of the kinetic barriers to subtilisin folding and to show how the prodomain overcomes these barriers. To address these questions, we have studied the bimolecular folding reaction of the subtilisin prodomain and a series of subtilisin mutants, which were designed to explore the steps in the folding reaction. Our analysis shows that inordinately slow folding of the mature form of subtilisin results from the accrued effects of two slow and sequential processes: (1) the formation of an unstable and topologically challenged intermediate and (2) the proline-limited isomerization of the intermediate to the native state. The low stability of nascent folding intermediates results in part from subtilisin's high dependence on metal binding for stability. Native subtilisin is thermodynamically unstable in the absence of bound metals. Because the two metal binding sites are formed late in folding, however, they contribute little to the stability of folding intermediates. The formation of productive folding intermediates is further hindered by the topological challenge of forming a left-handed crossover connection between beta-strands S2 and S3. This connection is critical to propagate the folding reaction. In the presence of the prodomain, folding proceeds through one major intermediate, which is stabilized by prodomain binding, independent of metal concentration and proline isomerization state. The prodomain also catalyzes the late proline isomerizations needed to form metal site B. Rate-limiting proline isomerization is common in protein folding, but its effect in slowing subtilisin folding is amplified because of the instability of the intermediate and an apparent need for simultaneous isomerization of multiple prolines in order to create metal site B. Thus, the kinetically controlled folding reaction of subtilisin, although unusual, is explained by the accrued effects of events found in other proteins.     

Topology and sequence in the folding of a TIM barrel protein: global analysis highlights partitioning between transient off-pathway and stable on-pathway folding intermediates in the complex folding mechanism of a (betaalpha)8 barrel of unknown function from B. subtilis

The relative contributions of chain topology and amino acid sequence in directing the folding of a (betaalpha)(8) TIM barrel protein of unknown function encoded by the Bacillus subtilis iolI gene (IOLI) were assessed by reversible urea denaturation and a combination of circular dichroism, fluorescence and time-resolved fluorescence anisotropy spectroscopy. The equilibrium reaction for IOLI involves, in addition to the native and unfolded species, a stable intermediate with significant secondary structure and stability and self-associated forms of both the native and intermediate states. Global kinetic analysis revealed that the unfolded state partitions between an off-pathway refolding intermediate and the on-pathway equilibrium intermediate early in folding. Comparisons with the folding mechanisms of two other TIM barrel proteins, indole-3-glycerol phosphate synthase from the thermophile Sulfolobus solfataricus (sIGPS) and the alpha subunit of Escherichia coli tryptophan synthase (alphaTS), reveal striking similarities that argue for a dominant role of the topology in both early and late events in folding. Sequence-specific effects are apparent in the magnitudes of the relaxation times and relative stabilities, in the presence of additional monomeric folding intermediates for alphaTS and sIGPS and in rate-limiting proline isomerization reactions for alphaTS.     

Proline cis-trans isomerization and protein folding

Proline cis-trans isomerization plays a key role in the rate-determining steps of protein folding. The energetic origin of this isomerization process is summarized, and the folding and unfolding of disulfide-intact bovine pancreatic ribonuclease A is used as an example to illustrate the kinetics and structural features of conformational changes from the heterogeneous unfolded state (consisting of cis and trans isomers of X-Pro peptide groups) to the native structure in which only one set of proline isomers is present.     

Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding

The refolding of chymotrypsin inhibitor 2 (CI2) is, at least, a triphasic process. The rate constants are 53 s-1 for the major phase (77% of the total amplitude) and 0.43 and 0.024 s-1 for the slower phases (23% of the total amplitude) at 25 degrees C and pH 6.3. The multiphase nature of the refolding reaction results from heterogeneity in the denatured state because of proline isomerization. The fast phase corresponds to the refolding of the fraction of protein that has all its prolines in a native trans conformation in the denatured state. It is not catalyzed by peptidyl-prolyl isomerase. The rate-limiting step of folding for the slower phases, however, is proline isomerization, and they are both catalyzed by peptidyl-prolyl isomerase. The slowest phase has properties consistent with a process involving proline isomerization in a denatured state. In particular, the activation enthalpy is large, 16 kcal mol-1 K-1, and the rate is independent of guanidinium chloride concentration ([GdnHCl]). In comparison, the intermediate phase shows properties consistent with a process involving proline isomerization in a partially structured state. The activation enthalpy is small, 8 kcal mol-1 K-1, and the rate has a strong dependence on [GdnHCl]. Temperature dependences of the rate constants for unfolding and for the fast refolding phase, both in the absence and in the presence of GdnHCl, were used to characterize the thermodynamic nature of the transition state and its relative exposure to solvent. The Eyring plot for unfolding is linear, indicating that there is relatively little change in heat capacity between native state and transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Folding and domain-domain interactions of the chaperone PapD measured by 19F NMR

The folding of the two-domain bacterial chaperone PapD has been studied to develop an understanding of the relationship between individual domain folding and the formation of domain-domain interactions. PapD contains six phenylalanine residues, four in the N-terminal domain and two in the C-terminal domain. To examine the folding properties of PapD, the protein was both uniformly and site-specifically labeled with p-fluoro-phenylalanine ((19)F-Phe) for (19)F NMR studies, in conjunction with those of circular dichroism and fluorescence. In equilibrium denaturation experiments monitored by (19)F NMR, the loss of (19)F-Phe native intensity for both the N- and C-terminal domains shows the same dependence on urea concentration. For the N-terminal domain the loss of native intensity is mirrored by the appearance of separate denatured resonances. For the C-terminal domain, which contains residues Phe 168 and Phe 205, intermediate as well as denatured resonances appear. These intermediate resonances persist at denaturant concentrations well beyond the loss of native resonance intensity and appear in kinetic refolding (19)F NMR experiments. In double-jump (19)F NMR experiments in which proline isomerization does not affect the refolding kinetics, the formation of domain-domain interactions is fast if the protein is denatured for only a short time. However, with increasing time of denaturation the native intensities of the N- and C-terminal domains decrease, and the denatured resonances of the N-terminal domain and the intermediate resonances of the C-terminal domain accumulate. The rate of loss of the N-terminal domain resonances is consistent with a cis to trans isomerization process, indicating that from an equilibrium denatured state the slow refolding of PapD is due to the trans to cis isomerization of one or both of the N-terminal cis proline residues. The data indicate that both the N- and C-terminal domains must fold into a native conformation prior to the formation of domain-domain interactions.     

Kinetic mechanism and catalysis of a native-state prolyl isomerization reaction

Folding of tendamistat is a rapid two-state process for the majority of the unfolded molecules. In fluorescence-monitored refolding kinetics about 8% of the unfolded molecules fold slowly (lambda=0.083s(-1)), limited by peptidyl-prolyl cis-trans isomerization. This is significantly less than expected from the presence of three trans prolyl-peptide bonds in the native state. In interrupted refolding experiments we detected an additional very slow folding reaction (lambda=0.008s(-1) at pH 2) with an amplitude of about 12%. This reaction is caused by the interconversion of a highly structured intermediate to native tendamistat. The intermediate has essentially native spectroscopic properties and about 2% of it remain populated in equilibrium after folding is complete. Catalysis by human cyclophilin 18 identifies this very slow reaction as a prolyl isomerization reaction. This shows that prolyl-isomerases are able to efficiently catalyze native state isomerization reactions, which allows them to influence biologically important regulatory conformational transitions. Folding kinetics of the proline variants P7A, P9A, P50A and P7A/P9A show that the very slow reaction is due to isomerization of the Glu6-Pro7 and Ala8-Pro9 peptide bonds, which are located in a region that makes strong backbone and side-chain interactions to both beta-sheets. In the P50A variant the very slow isomerization reaction is still present but native state heterogeneity is not observed any more, indicating a long-range destabilizing effect on the alternative native state relative to N. These results enable us to include all prolyl and non-prolyl peptide bond isomerization reactions in the folding mechanism of tendamistat and to characterize the kinetic mechanism and the energetics of a native-state prolyl isomerization reaction.     

Investigation of the folding pathway of the TEM-1 β-lactamase

The TEM-1 β-lactamase is a globular protein containing 12 proline residues. The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism. The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state. Thisstate is 5.2 ± 0.4 kcal/mol less stable than the native conformation and 5.7 ± 0.2 kcal/mol more stable than the fully denaturedprotein. This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the “molten globule” is discussed. Refolding kinetic experimentsrevealed the existence of a transient intermediate conformation between thethermodynamically stable intermediate and the native protein. This transient intermediate appears rapidly during the folding reaction. It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation. The final refolding step of the TEM-1 β-lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probablyreflect a very complex process kinetically limited by proline cis/transisomerization. © 1995 Wiley-Liss, Inc.     

Influence of the internal disulfide bridge on the folding pathway of the CL antibody domain

Disulfide bridges are one of the most important factors stabilizing the native structure of a protein. Whereas the basis for their stabilizing effect is well understood, their role in a protein folding reaction still seems to require further attention. We used the constant domain of the antibody light chain (C(L)), a representative of the ubiquitous immunoglobulin (Ig)-superfamily, to delineate the kinetic role of its single buried disulfide bridge. Independent of its redox state, the monomeric C(L) domain adopts a typical Ig-fold under native conditions and does not retain significant structural elements when unfolded. Interestingly, its folding pathway is strongly influenced by the disulfide bridge. The more stable oxidized protein folds via a highly structured on-pathway intermediate, whereas the destabilized reduced protein populates a misfolded off-pathway species on its way to the native state. In both cases, the formation of the intermediate species is shown to be independent of the isomerization state of the Tyr(141)-Pro(142) bond. Our results demonstrate that the internal disulfide bridge in an antibody domain restricts the folding pathway by bringing residues of the folding nucleus into proximity thus facilitating the way to the native state.     

The influence of proline isomerization and off-pathway intermediates on the folding mechanism of eukaryotic UMP/CMP Kinase

The globular 22-kDa protein UMP/CMP from Dictyostelium discoideum (UmpK) belongs to the family of nucleoside monophosphate (NMP) kinases. These enzymes not only show high sequence and structure similarities but also share the α/β-fold, a very common protein topology. We investigated the protein folding mechanism of UmpK as a representative for this ubiquitous enzyme class. Equilibrium stability towards urea and the unfolding and refolding kinetics were studied by means of fluorescence and far-UV CD spectroscopy. Although the unfolding can be described by a two-state process, folding kinetics are rather complex with four refolding phases that can be resolved and an additional burst phase. Moreover, two of these phases exhibit a pronounced rollover in the refolding limb that cannot be explained by aggregation. Whilst secondary structure formation is not observed in the burst phase reaction, folding to the native structure is strongly influenced by the slowest phase, since 30% of the α-helical CD signal is restored therein. This process can be assigned to proline isomerization and is strongly accelerated by the Escherichia coli peptidyl-prolyl isomerase trigger factor. The analysis of our single-mixing and double-mixing experiments suggests the occurrence of an off-pathway intermediate and an unproductive collapsed structure, which appear to be rate limiting for the folding of UmpK.     

Plasticity within the obligatory folding nucleus of an immunoglobulin-like domain

A number of β-sandwich immunoglobulin-like domains have been shown to fold using a set of structurally equivalent residues that form a folding nucleus deep within the core of the protein. Formation of this nucleus is sufficient to establish the complex Greek key topology of the native state. These nucleating residues are highly conserved within the immunoglobulin superfamily, but are less well conserved in the fibronectin type III (fnIII) superfamily, where the requirement is simply to have four interacting hydrophobic residues. However, there are rare examples where this nucleation pattern is absent. In this study, we have investigated the folding of a novel member of the fnIII superfamily whose nucleus appears to lack one of the four buried hydrophobic residues. We show that the folding mechanism is unaltered, but the folding nucleus has moved within the hydrophobic core.     

Cooperativity of a protein folding reaction probed at multiple chain positions by real-time 2D NMR spectroscopy

The refolding reaction of S54G/P55N ribonuclease T1 is a two-step process, where fast formation of a partly folded intermediate is followed by the slow reaction to the native state, limited by a trans --> cis isomerization of Pro39. The hydrodynamic radius of this kinetic folding intermediate was determined by real-time diffusion NMR spectroscopy. Its folding to the native state was monitored by a series of 128 very fast 2D (15)N-HMQC spectra, to observe the kinetics of 66 individual backbone amide probes. We find that the intermediate is as compact as the native protein with many native chemical shifts. All 66 analyzed amide probes follow the rate-limiting prolyl isomerization, which indicates that this cooperative refolding reaction is fully synchronized. The stability of the folding intermediate was determined from the protection factors of 45 amide protons derived from a competition between refolding and H/D exchange. The intermediate has already gained 40% of the Gibbs free energy of refolding with many protected amides in not-yet-native regions.     

Parallel channels and rate-limiting steps in complex protein folding reactions: prolyl isomerization and the alpha subunit of Trp synthase,a TIM barrel protein

A kinetic folding mechanism for the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli, involving four parallel channels with multiple native, intermediate and unfolded forms, has recently been proposed. The hypothesis that cis/trans isomerization of several Xaa-Pro peptide bonds is the source of the multiple folding channels was tested by measuring the sensitivity of the three rate-limiting phases (tau(1), tau(2), tau(3)) to catalysis by cyclophilin, a peptidyl-prolyl isomerase. Although the absence of catalysis for the tau(1) (fast) phase leaves its assignment ambiguous, our previous mutational analysis demonstrated its connection to the unique cis peptide bond preceding proline 28. The acceleration of the tau(2) (medium) and tau(3) (slow) refolding phases by cyclophilin demonstrated that cis/trans prolyl isomerization is also the source of these phases. A collection of proline mutants, which covered all of the remaining 18 trans proline residues of alphaTS, was constructed to obtain specific assignments for these phases. Almost all of the mutant proteins retained the complex equilibrium and kinetic folding properties of wild-type alphaTS; only the P217A, P217G and P261A mutations caused significant changes in the equilibrium free energy surface. Both the P78A and P96A mutations selectively eliminated the tau(1) folding phase, while the P217M and P261A mutations eliminated the tau(2) and tau(3) folding phases, respectively. The redundant assignment of the tau(1) phase to Pro28, Pro78 and Pro96 may reflect their mutual interactions in non-random structure in the unfolded state. The non-native cis isomers for Pro217 and Pro261 may destabilize an autonomous C-terminal folding unit, thereby giving rise to kinetically distinct unfolded forms. The nature of the preceding amino acid, the solvent exposure, or the participation in specific elements of secondary structure in the native state, in general, are not determinative of the proline residues whose isomerization reactions can limit folding.     

Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain

The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.     

Rapid folding of calcium-free subtilisin by a stabilized pro-domain mutant.

In vitro folding of mature subtilisin is extremely slow. The isolated pro-domain greatly accelerates in vitro folding of subtilisin in a bimolecular reaction whose product is a tight complex between folded subtilisin and folded pro-domain. In our studies of subtilisin, we are trying to answer two basic questions: why does subtilisin fold slowly without the pro-domain and what does the pro-domain do to accelerate the folding rate? To address these general questions, we are trying to characterize all the rate constants governing individual steps in the bimolecular folding reaction of pro-domain with subtilisin. Here, we report the results of a series of in vitro folding experiments using an engineered pro-domain mutant which is independently stable (proR9) and two calcium-free subtilisin mutants. The bimolecular folding reaction of subtilisin and proR9 occurs in two steps: an initial binding of proR9 to unfolded subtilisin, followed by isomerization of the initial complex into the native complex. The central findings are as follows. First, the independently stable proR9 folds subtilisin much faster than the predominantly unfolded wild-type pro-domain. Second, at micromolar concentrations of proR9, the subtilisin folding reaction becomes limited by the rate at which prolines in the unfolded state can isomerize to their native conformation. The simpliest mechanism which closely describes the data includes two denatured forms of subtilisin, which form the initial complex with proR9 at the same rate but which isomerize to the fully folded complex at much different rates. In this model, 77% of the subtilisin isomerizes to the native form slowly and the remaining 23% isomerizes more rapidly (1.5 s-1). The slow-folding population may be unfolded subtilisin with the trans form of proline 168, which must isomerize to the cis form during refolding. Third, in the absence of proline isomerization, the rate of subtilisin folding is rapid and at [proR9] 3 s-1. The implications of these results concerning why subtilisin folds slowly without the pro-domain are discussed.     

Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization

Unfolding and refolding kinetics of human FKBP12 C22A were monitored by fluorescence emission over a wide range of urea concentration in the presence and absence of protecting osmolytes glycerol, proline, sarcosine and trimethylamine-N-oxide (TMAO). Unfolding is well described by a mono-exponential process, while refolding required a minimum of two exponentials for an adequate fit throughout the urea concentration range considered. The bi-exponential behavior resulted from complex coupling between protein folding, and prolyl isomerization in the denatured state in which the urea-dependent rate constant for folding was greater than, equal to, and less than the rate constants for prolyl isomerization within the urea concentration range of zero to five molar. Amplitudes and the observed folding and unfolding rate constants were fitted to a reversible three-state model composed of two sequential steps involving the native state and a folding-competent denatured species thermodynamically linked to a folding-incompetent denatured species. Excellent agreement between thermodynamic parameters for FKBP12 C22A folding calculated from the kinetic parameters and those obtained directly from equilibrium denaturation assays provides strong support for the applicability of the mechanism, and provides evidence that FKBP12 C22A folding/unfolding is two-state, with prolyl isomer heterogeneity in the denatured ensemble. Despite the chemical diversity of the protecting osmolytes, they all exhibit the same kinetic behavior of increasing the rate constant of folding and decreasing the rate constant for unfolding. Osmolyte effects on folding/unfolding kinetics are readily explained in terms of principles established in understanding osmolyte effects on protein stability. These principles involve the osmophobic effect, which raises the Gibbs energy of the denatured state due to exposure of peptide backbone, thereby increasing the folding rate. This effect also plays a key role in decreasing the unfolding rate when, as is often the case, the activated complex exposes more backbone than is exposed in the native state.