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

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

Nonprolyl cis peptide bonds in unfolded proteins cause complex folding kinetics

Folding of tendamistat, an inhibitor of alpha-amylase, is a fast two-state process accompanied by two minor slow reactions, which were assigned to prolyl isomerization. In a proline-free variant, 5% of the molecules still fold slowly with a rate constant of 2.5 s(-1). This reaction is caused by a slow equilibrium between two populations of unfolded molecules. The time constant for this equilibration process, its sensitivity to LiCl and its temperature dependence identify it as a cis-trans isomerization of nonprolyl peptide bonds. Although nonprolyl peptide bonds have the cis conformation populating only approximately 0.15% in unfolded proteins, their large number generates a significant fraction of slow-folding molecules. This emphasizes that heterogeneous populations in an unfolded protein can induce complex folding kinetics on various time scales.     

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

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

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 mechanism of ketosteroid isomerase from Comamonas testosteroni

Ketosteroid isomerase (KSI) from Comamonas testosteroni is a homodimeric enzyme with 125 amino acids in each monomer catalyzing the allylic isomerization reaction at rates comparable to the diffusion limit. Kinetic analysis of KSI refolding has been carried out to understand its folding mechanism. The refolding process as monitored by fluorescence change revealed that the process consists of three steps with a unimolecular fast, a bimolecular intermediate, and most likely unimolecular slow phases. The fast refolding step might involve the formation of structured monomers with hydrophobic surfaces that seem to have a high binding capacity for the amphipathic dye 8-anilino-1-naphthalenesulfonate. During the refolding process, KSI also generated a state that can bind equilenin, a reaction intermediate analogue, at a very early stage. These observations suggest that the KSI folding might be driven by the formation of the apolar active-site cavity while exposing hydrophobic surfaces. Since the monomeric folding intermediate may contain more than 83% of the native secondary structures as revealed previously, it is nativelike taking on most of the properties of the native protein. Urea-dependence analysis of refolding revealed the existence of folding intermediates for both the intermediate and slow steps. These steps were accelerated by cyclophilin A, a prolyl isomerase, suggesting the involvement of a cis-trans isomerization as a rate-limiting step. Taken together, we suggest that KSI folds into a monomeric intermediate, which has nativelike secondary structure, an apolar active site, and exposed hydrophobic surface, followed by dimerization and prolyl isomerizations to complete the folding.     

Fast folding of Escherichia coli cyclophilin A: a hypothesis of a unique hydrophobic core with a phenylalanine cluster

Escherichia coli cyclophilin A, a 164 residue globular protein, shows fast and slow phases of refolding kinetics from the urea-induced unfolded state at pH 7.0. Given that the slow phases are independent of the denaturant concentration and may be rate-limited by cis/trans isomerizations of prolyl peptide bonds, the fast phase represents the true folding reaction. The extrapolation of the fast-phase rate constant to 0 M urea indicates that the folding reaction of cyclophilin A is extraordinarily fast and has about 700 s(-1) of the rate constant. Interrupted refolding experiments showed that the protein molecules formed in the fast phase had already been fully folded to the native state. This finding overthrows the accepted view that the fast folding is observed only in small proteins of fewer than 100 amino acid residues. Examination of the X-ray structure of cyclophilin A has shown that this protein has only one unique hydrophobic core (phenylalanine cluster) formed by evolutionarily conserved phenylalanine residues, and suggests that this architecture of the molecule may be responsible for the fast folding behavior.     

Folding mechanism of porcine ribonuclease

The kinetics of unfolding and refolding of porcine ribonuclease were investigated. The unfolded state of this protein was found to consist of a fast-refolding species (UF) and two slow-refolding species (UIS and UIIS). After the rapid collapse of the structure during the N (native)----UF unfolding reaction, UIS and UIIS are produced from UF by two independent slow isomerizations of the unfolded polypeptide chain, leading ultimately to a mixture of about 10% UF, 20% UIIS and 70% UIS molecules at equilibrium. This is at variance with all other ribonucleases investigated to date, which show a distribution of 20% UF, 60 to 70% UIIS and only 10 to 20% UIS. The two isomerizations of the unfolded porcine protein differ strongly in rate. The first process with tau = 250 seconds (10 degrees C) leads to an increase in the fluorescence of Tyr92 and was identified as cis in equilibrium trans isomerization of Pro93. At equilibrium, most unfolded molecules contain an incorrect trans Pro93. The second isomerization is much slower (tau = 1300 s at 10 degrees C) and leads to a predominance of the incorrect isomer as well. Like isomerization of Pro93, it is governed by an activation enthalpy of 22 kcal/mol (92 kJ/mol) and it was tentatively assigned to the Pro114-Pro115 sequence of porcine ribonuclease. Because of the wide separation in rate between the two reactions, molecules with an incorrect isomer only at Pro93 accumulate transiently after unfolding. These are the UIIS molecules. Most of them are finally converted to UIS by the 1300 second process. All molecules that have undergone this isomerization refold very slowly, i.e. the UIS molecules. The major fraction contains two incorrect isomers. A 1300 second isomerization after unfolding and a predominant very slow refolding reaction were observed only for the porcine protein. We suggest that these changes in the folding mechanism may be correlated with the presence of the Pro114-Pro115 sequence, which occurs only in porcine ribonuclease. The refolding pathway of porcine UIIS involves the rapid formation of a native-like intermediate with an incorrect trans Pro93 as was found previously for the bovine ribonuclease, where the UIIS species predominates in the unfolded state.     

Nature of the fast and slow refolding reactions of iron(III) cytochrome c

The fast and slow refolding reactions of iron(III) cytochrome c (Fe(III) cyt c), previously studied by Ikai et al. (Ikai, A., Fish, W. W., & Tanford, C. (1973) J. Mol. Biol. 73, 165--184), have been reinvestigated. The fast reaction has the major amplitude (78%) and is 100-fold faster than the slow reaction in these conditions (pH 7.2, 25 degrees C, 1.75 M guanidine hydrochloride). We show here that native cyt c is the product formed in the fast reaction as well as in the slow reaction. Two probes have been used to test for formation of native cyt c. absorbance in the 695-nm band and rate of reduction of by L-ascorbate. Different unfolded species (UF, US) give rise to the fast and slow refolding reactions, as shown both by refolding assays at different times after unfolding ("double-jump" experiments) and by the formation of native cyt c in each of the fast and slow refolding reactions. Thus the fast refolding reaction is UF leads to N and the slow refolding reaction is Us leads to N, where N is native cyt c, and there is a US in equilibrium UF equilibrium in unfolded cyt c. The results are consistent with the UF in equilibrium US reaction being proline isomerization, but this has not yet been tested in detail. Folding intermediates have been detected in both reactions. In the UF leads to N reaction, the Soret absorbance change precedes the recovery of the native 695-nm band spectrum, showing that Soret absorbance monitors the formation of a folding intermediate. In the US leads to N reaction an ascorbate-reducible intermediate has been found at an early stage in folding and the Soret absorbance change occurs together with the change at 695 nm as N is formed in the final stage of folding.     

Molecular Determinants of a Native-State Prolyl Isomerization

Prolyl cis/trans isomerizations determine the rates of many protein-folding reactions, and they can serve as molecular switches and timers. The energy required to shift the prolyl cis/trans equilibrium during these processes originates from conformational reactions that are linked structurally and energetically with prolyl isomerization. We used the N2 domain of the gene-3-protein of phage fd to elucidate how such an energetic linkage develops in the course of folding. The Asp160-Pro161 bond at the tip of a β hairpin of N2 is cis in the crystal structure, but in fact, it exists as a mixture of conformers in folded N2. During refolding, about 10 kJ mol^− 1 of conformational energy becomes available for a 75-fold shift of the cis/trans equilibrium constant at Pro161, from 7/93 in the unfolded to 90/10 in the folded form. We combined single- and double-mixing kinetic experiments with a mutational analysis to identify the structural origin of this proline shift energy and to elucidate the molecular path for the transfer of this energy to Pro161. It originates largely, if not entirely, from the two-stranded β sheet at the base of the Pro161 hairpin. The two strands improve their stabilizing interactions when Pro161 is cis, and this stabilization is propagated to Pro161, because the connector peptides between the β strands and Pro161 are native-like folded when Pro161 is cis. In the presence of a trans-Pro161, the connector peptides are locally unfolded, and thus, Pro161 is structurally and energetically uncoupled from the β sheet. Such interrelations between local folding and prolyl isomerization and the potential modulation by prolyl isomerases might also be used to break and reestablish slow communication pathways in proteins.     

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.     

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.     

Identification of proline residues responsible for the slow folding kinetics in pectate lyase C by mutagenesis

The folding mechanism of pectate lyase C (pelC) involves two slow phases that have been attributed to proline isomerization. To have a more detailed and complete understanding of the folding mechanism, experiments have been carried out to identify the prolyl-peptide bonds responsible for the slow kinetics. Site-directed mutagenesis has been used to mutate each of the prolines in pelC to alanine or valine. It has been determined that isomerization of the Leu219-Pro220 peptide bond is responsible for the slowest folding phase observed. The mutant P220A shows kinetic behavior that is identical to the wild-type protein except that the 46-s phase is eliminated. The Leu219-Pro220 peptide bond is cis in the native enzyme. An analysis of the free energy of unfolding of this mutant indicates that the mutation destabilizes the protein by about 4 kcal/mol. However, it appears that the major refolding pathways are unaltered. Further mutations were carried out in order to assign the peptide bond responsible for the 21-s folding phase in pelC. Mutation of the remaining 11 prolines, which are trans in the native enzyme, resulted in no significant changes in the kinetic folding behavior. The conclusion from these experiments is that the 21-s phase involves isomerization of more than one prolyl-peptide bond with similar activation energies.     

Folding mechanism of the CH2 antibody domain

The immunoglobulin C(H)2 domain is a simple model system suitable for the study of the folding of all-beta-proteins. Its structure consists of two beta-sheets forming a greek-key beta-barrel, which is stabilized by an internal disulfide bridge located in the hydrophobic core. Crystal structures of various antibodies suggest that the C(H)2 domains of the two heavy chains interact with their sugar moieties and form a homodimer. Here, we show that the isolated, unglycosylated C(H)2 domain is a monomeric protein. Equilibrium unfolding was a two-state process, and the conformational stability is remarkably low compared to other antibody domains. Folding kinetics of C(H)2 were found to consist of several phases. The reactions could be mapped to three parallel pathways, two of which are generated by prolyl isomerizations in the unfolded state. The slowest folding reaction, which was observed only after long-term denaturation, could be catalyzed by a prolyl isomerase. The majority of the unfolded molecules, however, folded more rapidly, on a time-scale of minutes. Presumably, these molecules also have to undergo prolyl isomerization before reaching the native state. In addition, we detected a small number of fast-folding molecules in which all proline residues appear to be in the correct conformation. On both prolyl isomerization limited pathways, the formation of partly structured intermediates could be observed.     

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.     

The two slow refolding processes of creatine kinase are catalyzed by cyclophilin

A burst phase occurs in the refolding kinetics of guanidine-denatured creatine kinase due to formation of an intermediate within the mixing dead time, with further refolding to the native state after the burst phase along a path following biphasic kinetics. In the presence of cyclophilin, the refolding rates of the two slow processes are accelerated and the values are proportional to the cyclophilin concentration. The activity of cyclophilin in accelerating the slow refolding processes of creatine kinase is totally inhibited by cyclosporin A, indicating that the cis—trans isomerization of the peptidyl—prolyl bonds is involved in the two slow refolding processes.     

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

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

Replacement of a cis proline simplifies the mechanism of ribonuclease T1 folding

The refolding of ribonuclease T1 is dominated by two major slow kinetic phases that show properties of proline isomerization reactions. We report here that the molecular origin of one of these processes is the trans----cis isomerization of the Ser54-Pro55 peptide bond, which is cis in the native protein but predominantly trans in unfolded ribonuclease T1. This is shown by a comparison of the wild type and a designed mutant protein where Ser54 and Pro55 were replaced by Gly54 and Asn55, respectively. This mutation leaves the thermal stability of the protein almost unchanged; however, in the absence of Pro55 one of the two slow phases in folding is abolished and the kinetic mechanism of refolding is dramatically simplified.     

Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.

The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.     

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.