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.     

Equilibrium and kinetic analysis of folding of ketosteroid isomerase from Comamonas testosteroni

Equilibrium and kinetic analyses have been carried out to elucidate the folding mechanism of homodimeric ketosteroid isomerase (KSI) from Comamonas testosteroni. The folding of KSI was reversible since the activity as well as the fluorescence and CD spectra was almost completely recovered after refolding. The equilibrium unfolding transitions monitored by fluorescence and CD measurements were almost coincident with each other, and the transition midpoint increased with increasing protein concentration. This suggests that the KSI folding follows a simple two-state mechanism consisting of native dimer and unfolded monomer without any thermodynamically stable intermediates. Sedimentation equilibrium analysis and size-exclusion chromatography of KSI at different urea concentrations supported the two-state model without any evidence of folded monomeric intermediates. Consistent with the two-state model, (1)H-(15)N HSQC spectra obtained for KSI in the unfolding transition region could be reproduced by a simple addition of the spectra of the native and the unfolded KSI. The KSI refolding kinetics as monitored by fluorescence intensity could be described as a fast first-order process followed by a second-order and a subsequent slow first-order processes with rate constants of 60 s(-)(1), 5.4 x 10(4) M(-)(1).s(-)(1), and 0.017 s(-)(1), respectively, at 0.62 M urea, suggesting that there may be a monomeric folding intermediate. After a burst phase that accounts for >83% of the total amplitude, the negative molar ellipticity at 225 nm increased slowly in a single phase at a rate comparable to that of the bimolecular intermediate step. The kinetics of activity recovery from the denatured state were markedly dependent upon the protein concentration, implying that the monomers are not fully active. Taken together, our results demonstrate that the dimerization induces KSI to fold into the complete structure and is crucial for maintaining the tertiary structure to perform efficient catalysis.     

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.     

Effect of multiple prolyl isomerization reactions on the stability and folding kinetics of the notch ankyrin domain: experiment and theory

Studies on the folding kinetics of the Notch ankyrin domain have demonstrated that the major refolding phase is slow, the minor refolding phase is limited by the isomerization of prolyl peptide bonds, and that unfolding is multiexponential. Here, we explore the relationship between prolyl isomerization and folding heterogeneity using a combination of experiment and simulation. Proline residues were replaced with alanine, both singly and in various combinations. These destabilizing substitutions combine to eliminate the minor refolding phase, although unfolding heterogeneity persists even when all seven proline residues are replaced. To test whether prolyl isomerization influences the major refolding phase, we modeled folding and prolyl isomerization as a system of sequential reactions. Simulations that use rate constants of the major folding phase of the Notch ankyrin domain to represent intrinsic folding indicate that even with seven prolyl isomerization reactions, only two significant phases should be observed, and that the fast observed phase provides a good approximation of the intrinsic folding in the absence of prolyl isomerization. These results indicate that the major refolding phase of the Notch ankyrin domain reflects an intrinsically slow folding transition, rather than coupling of fast folding events with slow prolyl isomerization steps. This is consistent with the observation that the single observed refolding phase of a construct in which all proline residues are replaced remains slow. Finally, the simulation fails to produce a second unfolding phase at high urea concentrations, indicating that prolyl isomerization does not play a role in the three-state mechanism that leads to this heterogeneity.     

Folding and assembly of dimeric human glutathione transferase A1-1

Glutathione transferases function as detoxification enzymes and ligand-binding proteins for many hydrophobic endogenous and xenobiotic compounds. The molecular mechanism of folding of urea-denatured homodimeric human glutathione transferase A1-1 (hGSTA1-1) was investigated. The kinetics of change were investigated using far-UV CD, Trp20 fluorescence, fluorescence-detected ANS binding, acrylamide quenching of Trp20 fluorescence, and catalytic reactivation. The very early stages of refolding (millisecond time range) involve the formation of structured monomers with native-like secondary structure and exposed hydrophobic surfaces that have a high binding capacity for the amphipathic dye ANS. Dimerization of the monomeric intermediates was detected using Trp fluorescence and occurs as fast and intermediate events. The intermediate event was distinguished from the fast event because it is limited by a preceding slow trans-to-cis isomerization reaction (optically silent in this study). At high concentrations of hFKBP, dimerization is not limited by the isomerization reaction, and only the fast event was detected. The fast (tau = 200 ms) and intermediate (tau = 2.5 s) events show similar urea-, temperature-, and ionic strength-dependent properties. The dimeric intermediate has a partially functional active site ( approximately 20%). Final reorganization to form the native tertiary and quaternary structures occurs during a slow, unimolecular, urea- and ionic strength-independent event. During this slow event (tau = 250 s), structural rearrangements at the domain interface occur at/near Trp20 and result in burial of Trp20. The slow event results in the regain of the fully functional dimer. The role of the C-terminus helix 9 (residues 210-221) as a structural determinant for this final event is proposed.     

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.     

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.     

Catalysis of protein folding by cyclophilins from different species.

Cyclophilins are a class of ubiquitous proteins with yet unknown function. They were originally discovered as the major binding proteins for the immunosuppressant cyclosporin A. The only known catalytic function of these proteins in vitro is the cis/trans isomerization of Xaa-Pro bonds in oligopeptides. This became clear after the discovery that bovine cyclophilin is identical with porcine prolyl isomerase. This enzyme accelerates slow, proline-limited steps in the refolding of several proteins. Here we demonstrate that the cyclophilins from man, pig, Neurospora crassa, Saccharomyces cerevisiae, and Escherichia coli are all active as prolyl isomerases and as catalysts of protein folding. This evolutionary conservation suggests that catalysis of prolyl peptide bond isomerization may be an important function of the cyclophilins. It could be related with de novo protein folding or be involved in regulatory processes. Catalysis of folding is very efficient in the presence of the high cellular concentrations of prolyl isomerase.     

New structural insights into the refolding of ribonuclease T1 as seen by time-resolved Fourier-transform infrared spectroscopy

To get new structural insights into different phases of the renaturation of ribonuclease T1 (RNase T1), the refolding of the thermally unfolded protein was initiated by rapid temperature jumps and detected by time-resolved Fourier-transform infrared spectroscopy. The characteristic spectral changes monitoring the formation of secondary structure and tertiary contacts were followed on a time scale of 10(-3) to 10(3) seconds permitting the characterization of medium and slow folding reactions. Additionally, structural information on the folding events that occurred within the experimental dead time was indirectly accessed by comparative analysis of kinetic and steady-state refolding data. At slightly destabilizing refolding temperatures of 45 degrees C, which is close to the unfolding transition region, no specific secondary or tertiary structure is formed within 180 ms. After this delay all infrared markers bands diagnostic for individual structural elements indicate a strongly cooperative and relatively fast folding, which is not complicated by the accumulation of intermediates. At strongly native folding temperatures of 20 degrees C, a folding species of RNase T1 is detected within the dead time, which already possesses significant amounts of antiparallel beta-sheets, turn structures, and to some degree tertiary contacts. The early formed secondary structure is supposed to comprise the core region of the five-stranded beta-sheet. Despite these nativelike characteristics the subsequent refolding events are strongly heterogeneous and slow. The refolding under strongly native conditions is completed by an extremely slow formation or rearrangement of a locally restricted beta-sheet region accompanied by the further consolidation of turns and denser backbone packing. It is proposed that these late events comprise the final packing of strand 1 (residues 40-42) of the five-stranded beta-sheet against the rest of this beta-sheet system within an otherwise nativelike environment. This conclusion was supported by the comparison of refolding of RNase T1 and its variant W59Y RNase T1 that enabled the assignment of these very late events to the trans-->cis isomerization reaction of the prolyl peptide bond preceding Pro-39.     

Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme

Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step.     

Folding and association of the antibody domain CH3: prolyl isomerization preceeds dimerization.

The simplest naturally occurring model system for studying immunoglobulin folding and assembly is the non-covalent homodimer formed by the C-terminal domains (CH3) of the heavy chains of IgG. Here, we describe the structure of recombinant CH3 dimer as determined by X-ray crystallography and an analysis of the folding pathway of this protein.Under conditions where prolyl isomerization does not contribute to the folding kinetics, formation of the beta-sandwich structure is the rate-limiting step. beta-Sheet formation of CH3 is a slow process, even compared to other antibody domains, while the subsequent association of the folded monomers is fast. After long-time denaturation, the majority of the unfolded CH3 molecules reaches the native state in two serial reactions, involving the re-isomerization of the Pro35-peptide bond to the cis configuration. The species with the wrong isomer accumulate as a monomeric intermediate. Importantly, the isomerization to the correct cis configuration is the prerequisite for dimerization of the CH3 domain. In contrast, in the Fab fragment of the same antibody, prolyl isomerization occurs after dimerization demonstrating that within one protein, comprised of highly homologous domains, both the kinetics of beta-sandwich formation and the stage at which prolyl isomerization occurs during the folding process can be completely different.     

Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c

Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, J. J., Jr., Muthukrishnan, K., & Nall, B. T. (1985) Biochemistry 24, 6680-6684]. The kinetics of absorbance-detected refolding of the alkaline form have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (tau 2) are detected, as in refolding to the native state at neutral pH. An additional kinetic phase (tau a) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparison to pH-jump measurements of the fully folded protein shows that phase tau a has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species--with a 695-nm absorbance band--are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus, (1) refolding to the alkaline form of iso-2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leading to native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple parallel-pathway folding of proline-free Staphylococcal nuclease

When a protein exhibits complex kinetics of refolding, we often ascribe the complexity to slow isomerization events in the denatured protein, such as cis/trans isomerization of peptidyl prolyl bonds. Does the complex folding kinetics arise only from this well-known reason? Here, we have investigated the refolding of a proline-free variant of staphylococcal nuclease by stopped-flow, double-jump techniques, to examine the folding reactions without the slow prolyl isomerizations. As a result, the protein folds into the native state along at least two accessible parallel pathways, starting from a macroscopically single denatured-state ensemble. The presence of intermediates on the individual folding pathways has revealed the existence of multiple parallel pathways, and is characterized by multi-exponential folding kinetics with a lag phase. Therefore, a "single" amino acid sequence can fold along the multiple parallel pathways. This observation in staphylococcal nuclease suggests that the multiple folding may be more general than we have expected, because the multiple parallel-pathway folding cannot be excluded from proteins that show simpler kinetics.     

Folding of ribonuclease T1. 2. Kinetic models for the folding and unfolding reactions

The slow refolding of ribonuclease T1 was investigated by different probes. Structural intermediates with secondary structure are formed early during refolding, as indicated by the rapid regain of a native-like circular dichroism spectrum in the amide region. This extensive structure formation is much faster than the slow steps of refolding, which are limited in rate by the reisomerization of incorrect proline isomers. The transient folding intermediates were also detected by unfolding assays, which make use of the reduced stability of folding intermediates relative to that of the native protein. The results of this and the preceding paper [Kiefhaber et al. (1990) Biochemistry (preceding paper in this issue)] were used to propose kinetic models for the unfolding and refolding of ribonuclease T1. The unfolding mechanism is based on the assumption that, after the structural unfolding step, the slow isomerizations of two X-Pro peptide bonds occur independently of each other in the denatured protein. At equilibrium a small amount of fast-folding species coexists with three slow-folding species: two with one incorrect proline isomer each and another, dominant species with both these prolines in the incorrect isomeric state. In the mechanism for refolding we assume that all slow-folding molecules can rapidly regain most of the secondary and part of the tertiary structure early in folding. Reisomerizations of incorrect proline peptide bonds constitute the slow, rate-limiting steps of refolding. A peculiar feature of the kinetic model for refolding is that the major unfolded species with two incorrect proline isomers can enter two alternative folding pathways, depending on which of the two reisomerizes first. The relative rates of reisomerization of the respective proline peptide bonds at the stage of the rapidly formed intermediate determine the choice of pathway. It is changed in the presence of prolyl isomerase, because this enzyme catalyzes these two isomerizations with different efficiency and consequently leads to a shift from the very slow to the intermediate refolding pathway.     

Insertion of a chaperone domain converts FKBP12 into a powerful catalyst of protein folding

The catalytic activity of human FKBP12 as a prolyl isomerase is high towards short peptides, but very low in proline-limited protein folding reactions. In contrast, the SlyD proteins, which are members of the FKBP family, are highly active as folding enzymes. They contain an extra "insert-in-flap" or IF domain near the prolyl isomerase active site. The excision of this domain did not affect the prolyl isomerase activity of SlyD from Escherichia coli towards short peptide substrates but abolished its catalytic activity in proline-limited protein folding reactions. The reciprocal insertion of the IF domain of SlyD into human FKBP12 increased its folding activity 200-fold and generated a folding catalyst that is more active than SlyD itself. The IF domain binds to refolding protein chains and thus functions as a chaperone module. A prolyl isomerase catalytic site and a separate chaperone site with an adapted affinity for refolding protein chains are the key elements for a productive coupling between the catalysis of prolyl isomerization and conformational folding in the enzymatic mechanisms of SlyD and other prolyl isomerases, such as trigger factor and FkpA.     

A nonessential role for Arg 55 in cyclophilin18 for catalysis of proline isomerization during protein folding

The protein folding process is often in vitro rate‐limited by slow cis‐trans proline isomerization steps. Importantly, the rate of this process in vivo is accelerated by prolyl isomerases (PPIases). The archetypal PPIase is the human cyclophilin 18 (Cyp18 or CypA), and Arg 55 has been demonstrated to play a crucial role when studying short peptide substrates in the catalytic action of Cyp18 by stabilizing the transition state of isomerization. However, in this study we show that a R55A mutant of Cyp18 is as efficient as the wild type to accelerate the refolding reaction of human carbonic anhydrase II (HCA II). Thus, it is evident that the active‐site located Arg 55 is not required for catalysis of the rate‐limiting prolyl cis‐trans isomerization steps during the folding of a protein substrate as HCA II. Nevertheless, catalysis of cis‐trans proline isomerization in HCA II occurs in the active‐site of Cyp18, since binding of the inhibitor cyclosporin A abolishes rate acceleration of the refolding reaction. Obviously, the catalytic mechanisms of Cyp18 can differ when acting upon a simple model peptide, four residues long, with easily accessible Pro residues compared with a large protein molecule undergoing folding with partly or completely buried Pro residues. In the latter case, the isomerization kinetics are significantly slower and simpler mechanistic factors such as desolvation and/or strain might operate during folding‐assisted catalysis, since binding to the hydrophobic active site is still a prerequisite for catalysis.     

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)     

Prolyl isomerases show low sequence specificity toward the residue following the proline

Prolyl isomerases catalyze the cis/trans isomerization of peptide bonds preceding proline. Previously, we had determined the specificity toward the residue before the proline for cyclophilin-, FKBP-, and parvulin-type prolyl isomerases by using proline-containing oligopeptides and refolding proteins as model substrates. Here, we report the specificities of members of these three prolyl isomerase families for the residue following the proline, again in short peptide and in refolding protein chains. Human cyclophilin 18 and parvulin 10 from Escherichia coli show high activity, but low specificity, with respect to the residue following the proline. Human FKBP12 prefers hydrophobic residues at this position in the peptide assays and shows a very low activity in the protein folding assays. This activity was strongly improved, and the sequence specificity was virtually eliminated after the insertion of a chaperone domain into the prolyl isomerase domain of human FKBP12.     

A partially folded intermediate species of the beta-sheet protein apo-pseudoazurin is trapped during proline-limited folding

The folding of apo-pseudoazurin, a 123-residue, predominantly beta-sheet protein with a complex Greek key topology, has been investigated using several biophysical techniques. Kinetic analysis of refolding using far- and near-ultraviolet circular dichroism (UV CD) shows that the protein folds slowly to the native state with rate constants of 0.04 and 0.03 min(-1), respectively, at pH 7.0 and at 15 degrees C. This process has an activation enthalpy of approximately 90 kJ/mole and is catalyzed by cyclophilin A, indicating that folding is limited by trans-cis proline isomerization, presumably around the Xaa-Pro 20 bond that is in the cis isomer in the native state. Before proline isomerization, an intermediate accumulates during folding. This species has a substantial signal in the far-UV CD, a nonnative signal in the near-UV CD, exposed hydrophobic surfaces (judged by 1-anilino naphthalenesulphonate binding), a noncooperative denaturation transition, and a dynamic structure (revealed by line broadening on the nuclear magnetic resonance time scale). We compare the properties of this intermediate with partially folded states of other proteins and discuss its role in folding of this complex Greek key protein.     

Real-time NMR studies on a transient folding intermediate of barstar.

The refolding of barstar, the intracellular inhibitor of barnase, is dominated by the slow formation of a cis peptidyl prolyl bond in the native protein. The triple mutant C40/82A P27A in which two cysteine residues and one trans proline were replaced by alanine was used as model system to investigate the kinetics and structural consequences of the trans/cis interconversion of Pro48. One- and two-dimensional real-time NMR spectroscopy was used to follow the trans/cis interconversion after folding was initiated by rapid dilution of the urea denatured protein. Series of 1H, 15N HSQC spectra acquired with and without the addition of peptidyl prolyl isomerase unambiguously revealed the accumulation of a transient trans-Pro48 intermediate within the dead time of the experiment. Subtle chemical shift differences between the native state and the intermediate spectra indicate that the intermediate is predominantly native-like with a local rearrangement in the Pro48 loop and in the beta-sheet region including residues Tyr47, Ala82, Thr85, and Val50.