The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 β-lactamase is the trans to cis isomerization of a non-proline peptide bond

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 β-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166–Thr167 peptide bond, like the Glu166–Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans→cis isomerization of the Glu166–Thr167 peptide bond. © 1996 John Wiley & Sons, Inc.     

Proline peptide isomerization and protein folding

The unfolding-refolding of proteins is a cooperative process and, as judged by equilibrium properties, occurs in one step involving the native,N, and the unfoldedU, conformational states. Kinetic studies have shown that the denatured protein exists as a mixture of slow-(U)_Sand fast-(U)_Frefolding forms produced by proline peptidecis-trans isomerization. Proline residues inU _Fare in the same configuration as in the native protein while they are in non-native configuration inU _S. For protein folding to occur quicklyU _Smust be converted intoU _F. The fact that the equilibrium and kinetic properties of are the same as those found for prolinecis-trans isomerization taken together with the absence of slow phase in the kinetics of refolding of a protein devoid of proline, support this view. However, the absence of a linear correlation between half-time of reactivation of denatured enzymes and their proline-contents, as well as the dissimilarities in the kinetic properties of in unfolding and refolding experiments are not consistent with the model. Conformational energy calculation and experimental results on refolding of proteins suggest that some proline residues are non-essential. They will not block protein folding even in wrong isomeric form. The native-like folded structure with incorrect proline isomers will serve as intermediate state(s) in which these prolines will more readily isomerize to the correct isomeric form. The picture becomes more complex when one considers the consequence ofcis-trans isomerism of non-proline residues on protein folding.     

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.     

Structural mechanism governing cis and trans isomeric states and an intramolecular switch for cis/trans isomerization of a non-proline peptide bond observed in crystal structures of scorpion toxins

Non-proline cis peptide bonds have been observed in numerous protein crystal structures even though the energetic barrier to this conformation is significant and no non-prolyl-cis/trans-isomerase has been identified to date. While some external factors, such as metal binding or co-factor interaction, have been identified that appear to induce cis/trans isomerization of non-proline peptide bonds, the intrinsic structural basis for their existence and the mechanism governing cis/trans isomerization in proteins remains poorly understood. Here, we report the crystal structure of a newly isolated neurotoxin, the scorpion alpha-like toxin Buthus martensii Karsch (BmK) M7, at 1.4A resolution. BmK M7 crystallizes as a dimer in which the identical non-proline peptide bond between residues 9 and 10 exists either in the cis conformation or as a mixture of cis and trans conformations in either monomer. We also determined the crystal structures of several mutants of BmK M1, a representative scorpion alpha-like toxin that contains an identical non-proline cis peptide bond as that observed in BmK M7, in which residues within or neighboring the cis peptide bond were altered. Substitution of an aspartic acid residue for lysine at residue 8 in the BmK M1 (K8D) mutant converted the cis form of the non-proline peptide bond 9-10 into the trans form, revealing an intramolecular switch for cis-to-trans isomerization. Cis/trans interconversion of the switch residue at position 8 appears to be sequence-dependent as the peptide bond between residues 9 and 10 retains its wild-type cis conformation in the BmK M1 (K8Q) mutant structure. The structural interconversion of the isomeric states of the BmK M1 non-proline cis peptide bond may relate to the conversion of the scorpion alpha-toxins subgroups.     

A cis-prolyl peptide bond isomerization dominates the folding of the alpha subunit of Trp synthase,a TIM barrel protein

The cis/trans isomerization of prolyl peptide bonds has been suggested to dominate the folding of the alpha subunit of tryptophan synthase from Escherichia coli (alphaTS). To test the role of the unique cis isomer between Asp27 and Pro28, the folding properties of P28A, P28G and G(3)P28G, a three-glycine insertion mutant between Asp27 and Gly28, were investigated using urea as a denaturant. Circular dichroism analysis demonstrated that none of the mutations perturb the secondary structure significantly, although the aromatic side-chain packing is altered for P28A and P28G. All three mutant proteins inherited the three-state thermodynamic behavior observed in wild-type alphaTS, ensuring that the fundamental features of the energy surface are intact. Kinetic studies showed that neither alanine nor glycine substitutions at Pro28 results in the elimination of any slow-refolding phases. By contrast, the G(3)P28G mutant eliminates the fastest of the slow-refolding phases and one of the two unfolding phases. Double-jump experiments on G(3)P28G confirm the assignment of the missing refolding phase to the isomerization of the Asp27-Pro28 peptide bond. These results imply that the local stability conveyed by the tight, overlapping turns containing the cis peptide bond is sufficient to favor the cis isomer for several non-prolyl residues. The free energy required to drive the isomerization reaction is provided by the formation of the stable intermediate, demonstrating that the acquisition of structure and stability is required to induce subsequent rate-limiting steps in the folding of alphaTS.     

A hydrophobic cluster forms early in the folding of dihydrofolate reductase

The rapid kinetic phase that leads from unfolded species to transient folding intermediates in dihydrofolate reductase from Escherichia coli was examined by site-directed mutagenesis and by physicochemical means. The absence of this fluorescence-detected phase in the refolding of the Trp-74Phe mutant protein strongly implies that this early phase in refolding can be assigned to just one of the five Trp residues in the protein, Trp-74. In addition, water-soluble fluorescence quenching agents, iodide and cesium, have a much less significant effect on this early step in refolding than on the slower phases that lead to native and native-like conformers. These and other data imply that an important early event in the folding of dihydrofolate reductase is the formation of a hydrophobic cluster which protects Trp-74 from solvent.     

Dynamics of hydrogen bond desolvation in protein folding

As proteins fold, a progressive structuring, immobilization and eventual exclusion of water surrounding backbone hydrogen bonds takes place. This process turns hydrogen bonds into major determinants of the folding pathway and compensates for the penalty of desolvation of the backbone polar groups. Taken as an average over all hydrogen bonds in a native fold, this extent of protection is found to be nearly ubiquitous. It is dynamically crucial, determining a constraint in the long-time limit behavior of coarse-grained ab initio simulations. Furthermore, an examination of one of the longest available (1micros) all-atom simulations with explicit solvent reveals that this average extent of protection is a constant of motion for the folding trajectory. We propose how such a stabilization is best achieved by clustering five hydrophobes around the backbone hydrogen bonds, an arrangement that yields the optimal stabilization. Our results support and clarify the view that hydrophobic surface burial should be commensurate with hydrogen-bond formation and enable us to define a basic desolvation motif inherent to structure and folding dynamics.     

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.     

Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase

The unfolded state of a protein is an ensemble of a large number of conformations ranging from fully extended to compact structures. To investigate the effects of the difference in the unfolded-state ensemble on protein folding, we have studied the structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond, and compared the results with those of disulfide-reduced "linear" DHFR. Equilibrium studies by circular dichroism, difference absorption spectra, solution X-ray scattering, and size-exclusion chromatography show that whereas the native structures of both proteins are essentially the same, the unfolded state of circular DHFR adopts more compact conformations than the unfolded state of the linear form, even with the absence of secondary structure. Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization. Kinetic refolding measurements by stopped-flow circular dichroism and fluorescence show that under the native conditions both proteins accumulate a burst-phase intermediate having the same structures and both fold by the same complex folding mechanism with the same folding rates. Thus, the effects of the difference in the unfolded state of circular and linear DHFRs on the refolding reaction are not observed after the formation of the intermediate. This suggests that for the proteins with close termini in the native structure, early compaction of a protein molecule to form a specific folding intermediate with the N and C-terminal regions in close proximity is a crucial event in folding. If there is an enhancement in the folding reflecting the reduction in the breadth of the unfolded-state ensemble for circular DHFR, this acceleration must occur in the sub-millisecond time-range.     

Correlation of folding kinetics with the number and isomerization states of prolines in three homologous proteins of the RNase family

Several studies attribute the slower phases in protein folding to prolyl isomerizations, and several others do not. A correlation exists between the number of prolines in a protein and the complexity of the mechanism with which it folds. In this study, we have demonstrated a direct correlation between the number of cis-prolyl bonds in a native protein and the complexity with which it folds via slower phases by studying the folding of three structurally homologous proteins of the ribonuclease family, namely RNase A, onconase and angiogenin, which differ in the number and isomerization states of their proline residues.     

Microsecond subdomain folding in dihydrofolate reductase

The characterization of microsecond dynamics in the folding of multisubdomain proteins has been a major challenge in understanding their often complex folding mechanisms. Using a continuous-flow mixing device coupled with fluorescence lifetime detection, we report the microsecond folding dynamics of dihydrofolate reductase (DHFR), a two-subdomain α/β/α sandwich protein known to begin folding in this time range. The global dimensions of early intermediates were monitored by Förster resonance energy transfer, and the dynamic properties of the local Trp environments were monitored by fluorescence lifetime detection. We found that substantial collapse occurs in both the locally connected adenosine binding subdomain and the discontinuous loop subdomain within 35 μs of initiation of folding from the urea unfolded state. During the fastest observable ∼ 550 μs phase, the discontinuous loop subdomain further contracts, concomitant with the burial of Trp residue(s), as both subdomains achieve a similar degree of compactness. Taken together with previous studies in the millisecond time range, a hierarchical assembly of DHFR—in which each subdomain independently folds, subsequently docks, and then anneals into the native conformation after an initial heterogeneous global collapse—emerges. The progressive acquisition of structure, beginning with a continuously connected subdomain and spreading to distal regions, shows that chain entropy is a significant organizing principle in the folding of multisubdomain proteins and single-domain proteins. Subdomain folding also provides a rationale for the complex kinetics often observed.     

Probing the interactions between the folding elements early in the folding of Escherichia coli dihydrofolate reductase by systematic sequence perturbation analysis

One of the necessary conditions for a protein to be foldable is the presence of a complete set of folding elements (FEs) that are short contiguous peptide segments distributed over an amino acid sequence. Previous studies indicated the FE assembly model of protein folding, in which the FEs interact with each other and coalesce to form an intermediate(s) early in the folding reaction. This suggests that a clue to the understanding of the determinants of protein foldability can be found by investigating how the FEs interact with each other early in the folding and thereby elucidating roles of the FEs in protein folding. To reveal the formation process of FE-FE interactions, we studied the early folding events of Escherichia coli dihydrofolate reductase (DHFR) utilizing systematic sequence perturbation analysis. Here, systematic single amino acid substitutions were introduced inside of the FEs (W30X in FE2, V40X in FE3, N59X in FE4, and I155X in FE10; X refers to various amino acid residues), and their kinetic refolding reactions were measured by stopped-flow circular dichroism and fluorescence. We show that the interactions around Trp30 and Ile155 are formed in the burst phase intermediate, while those around Val40 and Asn59 are formed in the transition state of the subsequent folding phase (tau5-phase) and in much later processes, respectively. These and previous results suggest that FE2 and FE10, and also FE1 and FE7, involved in the loop subdomain of DHFR, interact with each other within a millisecond time range, while the stable FE3-FE4 interactions are formed in the later processes. This may highlight the important roles of the FEs mainly inside of the loop subdomain in formation of the burst phase intermediate having a hydrophobic cluster and native-like overall topology and in acquisition of the foldability of DHFR.     

Testing the relationship between foldability and the early folding events of dihydrofolate reductase from Escherichia coli

A "folding element" is a contiguous peptide segment crucial for a protein to be foldable and is a new concept that could assist in our understanding of the protein-folding problem. It is known that the presence of the complete set of folding elements of dihydrofolate reductase (DHFR) from Escherichia coli is essential for the protein to be foldable. Since almost all of the amino acid residues known to be involved in the early folding events of DHFR are located within the folding elements, a close relationship between the folding elements and early folding events is hypothesized. In order to test this hypothesis, we have investigated whether or not the early folding events are preserved in circular permutants and topological mutants of DHFR, in which the order of the folding elements is changed but the complete set of folding elements is present. The stopped-flow circular dichroism (CD) measurements show that the CD spectra at the early stages of folding are similar among the mutants and the wild-type DHFR, indicating that the presence of the complete set of folding elements is sufficient to preserve the early folding events. We have further examined whether or not sequence perturbation on the folding elements by a single amino acid substitution affects the early folding events of DHFR. The results show that the amino acid substitutions inside of the folding elements can affect the burst-phase CD spectra, whereas the substitutions outside do not. Taken together, these results indicate that the above hypothesis is true, suggesting a close relationship between the foldability of a protein and the early folding events. We propose that the folding elements interact with each other and coalesce to form a productive intermediate(s) early in the folding, and these early folding events are important for a protein to be foldable.     

Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.

The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.     

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Δ52 in an αC–βC loop. Double deletion mutants showed that the loop αC–βC has nonadditive couplings with the βF–βG and βG–βH loops regarding stability. Single deletion to the loops αC–βC or βC–βD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.     

Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate.

The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans----cis reisomerization of the Tyr-38-Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans----cis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate rate-limiting steps by trapping partially folded structures.     

PBOND： Web Server for the Prediction of Proline and Non-Proline cis / trans Isomerization

PBOND is a web server that predicts the conformation of the peptide bond between any two amino acids. PBOND classifies the peptide bonds into one out of four classes, namely cis imide (cis-Pro), cis amide (cis-nonPro), trans imide (trans-Pro) and trans amide (trans-nonPro). Moreover, for every prediction a reliability index is computed. The underlying structure of the server consists of three stages: (1) feature extraction, (2) feature selection and (3) peptide bond clas- sification. PBOND can handle both s...     

Structure and heterogeneity of the one- and two-disulfide folding intermediates of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a factor Xa-specific inhibitor and is structurally homologous to bovine pancreatic trypsin inhibitor (BPTI). The fully reduced TAP refolds spontaneously to form the native structure under a wide variation of redox buffers. The folding intermediates of TAP consist of at least 22 fractions of one-disulfide, two-disulfide, and three-disulfide scrambled isomers. Three species of well-populated one- and two-disulfide intermediates were isolated and structurally characterized. The predominant one-disulfide species contains TAP-(Cys33—Cys55). Two major two-disulfide isomers were TAP-(Cys33—Cys55, Cys15—Cys39) and TAP-(Cys33—Cys55, Cys5—Cys39). Both Cys33—Cys55 and Cys15—Cys39 are native disulfides of TAP. These three species are structural counterparts of BPTI-(Cys30—Cys51), BPTI-(Cys30—Cys51, Cys14—Cys38), and BPTI-(Cys30—Cys51,Cys5—Cys38), which have been shown to be the major intermediates of BPTI folding. In addition, time-course-trapped folding intermediates of TAP, consisting of about 47% one-disulfide species and 30% two-disulfide species, were collectively digested with thermolysin, and fragmented peptides were analyzed by Edman sequencing and mass spectrometry in order to characterize the disulfide-containing peptides. Among the 15 possible single-disulfide pairings of TAP, 10 (2 native and 8 nonnative) were found as structural components of its one- and two-disulfide folding intermediates. The results demonstrate that the major folding intermediates of TAP bear structural homology to those of BPTI. However, the folding pathway of TAP differs from that of BPTI by (a) a higher degree of heterogeneity of one- and two-disulfide intermediates and (b) the presence of three-disulfide scrambled isomers as folding intermediates. Mechanism(s) that may account for these diversities are proposed and discussed.     

Extending the folding nucleus of ubiquitin with an independently folding beta-hairpin finger: hurdles to rapid folding arising from the stabilisation of local interactions

The N-terminal beta-hairpin sequence of ubiquitin has been implicated as a folding nucleation site. To extend and stabilise the ubiquitin folding nucleus, we have inserted an autonomously folding 14-residue peptide sequence beta4 which in isolation forms a highly populated beta-hairpin (>70%) stabilised by local interactions. NMR structural analysis of the ubiquitin mutant (Ubeta4) shows that the hairpin finger is fully structured and stabilises ubiquitin by approximately 8kJmol(-1). Protein engineering and kinetic (phi(F)-value) analysis of a series of Ubeta4 mutants shows that the hairpin extension of Ubeta4 is also significantly populated in the transition state (phi(F)-values >0.7) and has the effect of templating the formation of native contacts in the folding nucleus of ubiquitin. However, at low denaturant concentrations the chevron plot of Ubeta4 shows a small deviation from linearity (roll-over effect), indicative of the population of a compact collapsed state, which appears to arise from over-stabilisation of local interactions. Destabilising mutations within the native hairpin sequence and within the engineered hairpin extension, but not elsewhere, eliminate this non-linearity and restore apparent two-state behaviour. The pitfall to stabilising local interactions is to present hurdles to the rapid and efficient folding of small proteins down a smooth folding funnel by trapping partially folded or misfolded states that must unfold or rearrange before refolding.     

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.