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.     

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.     

Protein Disulfide-Isomerase Interacts with a Substrate Protein at All Stages along Its Folding Pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a K_d of ca. 10⁻⁵ M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI''s interaction with a partly-folded protein, and the first to analyze this folding catalyst''s changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility.     

Kinetic analysis of the folding and unfolding of a mutant form of bovine pancreatic trypsin inhibitor lacking the cysteine-14 and -38 thiols

The kinetics of the disulfide-coupled unfolding-refolding transition of a mutant form of bovine pancreatic trypsin inhibitor (BPTI) lacking Cys-14 and -38 were measured and compared to previous results for the wild-type protein and other modified forms. The altered cysteines, which were changed to serine in the mutant protein, are normally paired in a disulfide in the native protein but from disulfides with Cys-5 in two-disulfide kinetic intermediates during folding. Although the mutant protein could fold efficiently, the kinetics of both folding and unfolding were altered, reflecting the roles of these cysteines in the two-disulfide intermediates with "wrong" disulfides. The intramolecular rate constant for the formation of the second disulfide of the native mutant protein was more than 10(3)-fold lower than that for the formation of a second disulfide during the refolding of the wild-type protein. The observed rate of unfolding of the mutant protein was also lower than that of the wild-type protein, demonstrating that the altered cysteines are involved in the intramolecular rearrangements that are the rate-determining step in the unfolding of the wild-type protein. These results confirm the previous conclusion [Creighton, T.E. (1977) J. Mol. Biol. 113, 275-293] that the energetically preferred pathway for folding and unfolding of BPTI includes intramolecular rearrangements of intermediates in which Cys-14 and -38 are paired in disulfides not present in the native protein. The present results are also consistent with other, less detailed, studies with similar mutants lacking Cys-14 and -38 [Marks, C.B., Naderi, H., Kosen, P.A., Kuntz, I.D., & Anderson, S. (1987) Science (Washington, D.C.) 235, 1370-1371].     

Proline isomerism in the protein folding of ribonuclease A

The guanidinium chloride-unfolded state of ribonuclease A was found to be an equilibrium mixture of slow- and fast-refolding forms of the protein chain, as has been suggested. Both forms appear to have the same spectroscopic observables as judged by the relative changes in fluorescence emission and polarization. The equilibrium between them is thermally dependent, with deltaHapp equal to -1.4 kcal/mol. The activation energy Ea is equal to 18 kcal/mol. These findings are consistent with the proposal that cis-trans isomerism of peptide bonds that are NH2-terminal to proline residues is responsible for the slow phase of RNase A refolding. However, the actual dependence of the magnitude of the slow reaction on initial, prefolding temperature cannot be explained by a model in which the proline configurations of the fast refolding form must be identical to those of the native protein, as has been suggested. Instead, the data reveal that, although the native structure of RNase A contains two cis prolines, cis isomers need not be present in the fast-refolding form in order for folding to occur.     

Limited conformational space for early-stage protein folding simulation

MOTIVATION: The problem of early-stage protein folding is critical for protein structure prediction. The model presented introduces a common definition of protein structures which may be treated as the possible in silico early-stage form of the polypeptide chain. Limitation of the conformational space to the ellipse path on the Ramachandran map was tested as a possible sub-space to represent the early-stage structure for simulation of protein folding. The proposed conformational sub-space was developed on the basis of the backbone conformation, with side-chain interactions excluded. RESULTS: The ellipse-path-limited conformation of BPTI was created using the criterion of shortest distance between Phi, Psi angles in native form of protein and the Phi, Psi angles belonging to the ellipse. No knots were observed in the structure created according to ellipse-path conformational sub-space. The energy minimization procedure applied to ellipse-path derived conformation directed structural changes toward the native form of the protein with SS-bonds system introduced to the procedure. AVAILABILITY: Program 'Ellipse' to create the ellipse-path derived structure available on request: myroterm@cyf-kr.edu.pl     

Proline isomerization in bovine pancreatic ribonuclease A. 2. Folding conditions

The kinetics of cis-trans isomerization of individual X-Pro peptide groups is used to study the backbone dynamics of bovine pancreatic ribonuclease A (RNase A). We previously developed and validated a fluorescence method for monitoring the cis-trans isomerization of the Tyr92-Pro93 and Asn113-Pro114 peptide groups of RNase A under unfolding conditions [Juminaga, D., Wedemeyer, W. J., and Scheraga, H. A. (1998) Biochemistry 37, 11614-11620]. The essence of this method is to introduce a fluorescent residue (Tyr or Trp) in a position adjacent to the isomerizing proline (if one is not already present) and to eliminate the fluorescence of other such residues adjacent to prolines by mutating them to phenylalanine. Here, we extend this method to observe the cis-trans isomerization of these peptide groups under folding conditions using two site-directed mutants (Y92F and Y115F) of RNase A. Both isomerizations decelerate with increasing concentrations of GdnHCl, with nearly identical m values (1.11 and 1.19 M(-1), respectively) and extrapolated zero-GdnHCl time constants (42 and 32 s, respectively); by contrast, under unfolding conditions, the cis-trans isomerizations of both Pro93 and Pro114 are independent of GdnHCl concentration. Remarkably, the isomerization rates under folding conditions at GdnHCl concentrations above 1 M are significantly slower than those measured under unfolding conditions. The temperature dependence of the Pro114 isomerization under folding conditions is also unusual; whereas Pro93 exhibits an activation energy typical of proline isomerization (19.4 kcal/mol), Pro114 exhibits a sharply reduced activation energy of 5.7 kcal/mol. A structurally plausible model accounts for these results and, in particular, shows that folding conditions strongly accelerate the cis-trans isomerization of both peptide groups to their native cis conformation, suggesting the presence of flickering local structure in their beta-hairpins.     

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.     

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.     

The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein

We have determined the three-dimensional structure of a two-disulfide intermediate (Cys(8)-Cys(20), Cys(14)-Cys(26)) on the oxidative folding pathway of the cyclotide MCoTI-II. Cyclotides have a range of bioactivities and, because of their exceptional stability, have been proposed as potential molecular scaffolds for drug design applications. The three-dimensional structure of the stable two-disulfide intermediate shows for the most part identical secondary and tertiary structure to the native state. The only exception is a flexible loop, which is collapsed onto the protein core in the native state, whereas in the intermediate it is more loosely associated with the remainder of the protein. The results suggest that the native fold of the peptide does not represent the free energy minimum in the absence of the Cys(1)-Cys(18) disulfide bridge and that although there is not a large energy barrier, the peptide must transiently adopt an energetically unfavorable state before the final disulfide can form.     

Structural prediction of the beta-domain of metallothionein-3 by molecular dynamics simulation

The beta-domain of metallothionein-3 (MT3) has been reported to be crucial to the neuron growth inhibitory bioactivity. Little detailed three-dimensional structural information is available to present a reliable basis for elucidation on structure-property-function relationships of this unique protein by experimental techniques. So, molecular dynamics simulation is adopted to study the structure of beta-domain of MT3. In this article, a 3D structural model of beta-domain of MT3 was generated. The molecular simulations provide detailed protein structural information of MT3. As compared with MT2, we found a characteristic conformation formed in the fragment (residue 1-13) at the N-terminus of MT3 owing to the constraint induced by 5TCPCP9, in which Pro7 and Pro9 residues are on the same side of the protein, both facing outward and the two 5-member rings of prolines are arranged almost in parallel, while Thr5 is on the opposite side. Thr5 in MT3 is also found to make the first four residues relatively far from the fragment (residue 23-26) as compared with MT2. The simulated structure of beta-domain of MT3 is looser than that of MT2. The higher energy of MT3 than that of MT2 calculated supports these conclusions. Simulation on the four isomer arising from the cis- or trans-configuration of 6CPCP9 show that the trans-/trans-isomer is energetic favorable. The partially unfolding structure of beta-domain of MT3 is also simulated and the results show the influence of 6CPCP9 sequence on the correct folding of this domain. The correlations between the bioactivity of MT3 and the simulated structure as well as the folding of beta-domain of MT3 are discussed based on our simulation and previous results.     

Secretion incompetence of bovine pancreatic trypsin inhibitor expressed in Escherichia coli.

There is increasing evidence that protein folding and protein export are competing processes in prokaryotic cells. Virtually all secretion studies reported to date, however, have employed proteins that are relatively uncharacterized in terms of their folding behavior and three-dimensional structure. In contrast, the structural and biochemical parameters governing the folding of bovine pancreatic trypsin inhibitor (BPTI) and several of its mutants have been studied intensively. We therefore undertook a study of the secretion behavior in Escherichia coli of recombinant BPTI and its mutants. Wild-type BPTI and two well-characterized folding mutants (C14A, C38A)BPTI and (C30A, C51A)BPTI (missing the 14-38 and 30-51 disulfide bonds, respectively), were investigated by analyzing their expression fused to an E. coli signal sequence or to two synthetic IgG-binding domains of staphylococcal protein A. Both disulfide mutants are destabilized relative to wild-type BPTI and exhibit markedly altered folding kinetics: one (C14A, C38A) folds more slowly than wild-type BPTI and the other (C30A, C51A) unfolds more rapidly. Both mutants were observed to be exported 3-10 times more efficiently than the wild-type molecule. Moreover, the levels of unprocessed preprotein in the cytoplasm were severalfold higher for the wild-type fusion than for the fusion to the two folding mutants. Intracellular degradation of the BPTI moiety was also observed. These results are consistent with traffic of intracellular BPTI preproteins on at least three routes along the secretory pathway: (a) facile secretion of unfolded material, (b) intracellular folding leading to secretion blockage, and (c) degradation followed by export of truncated molecules. A novel feature of these findings is the implication that disulfide bonds can form in the bacterial cytoplasm and lead to secretion incompetence.     

Knots in rings. The circular knotted protein Momordica cochinchinensis trypsin inhibitor-II folds via a stable two-disulfide intermediate

The aim of this work was to elucidate the oxidative folding mechanism of the macrocyclic cystine knot protein MCoTI-II. We aimed to investigate how the six-cysteine residues distributed on the circular backbone of the reduced unfolded peptide recognize their correct partner and join up to form a complex cystine-knotted topology. To answer this question, we studied the oxidative folding of the naturally occurring peptide using a range of spectroscopic methods. For both oxidative folding and reductive unfolding, the same disulfide intermediate species was prevalent and was characterized to be a native-like two-disulfide intermediate in which the Cys1-Cys18 disulfide bond was absent. Overall, the folding pathway of this head-to-tail cyclized protein was found to be similar to that of linear cystine knot proteins from the squash family of trypsin inhibitors. However, the pathway differs in an important way from that of the cyclotide kalata B1, in that the equivalent two-disulfide intermediate in that case is not a direct precursor of the native protein. The size of the embedded ring within the cystine knot motif appears to play a crucial role in the folding pathway. Larger rings contribute to the independence of disulfides and favor an on-pathway native-like intermediate that has a smaller energy barrier to cross to form the native fold. The fact that macrocyclic proteins are readily able to fold to a complex knotted structure in vitro in the absence of chaperones makes them suitable as protein engineering scaffolds that have remarkable stability.     

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.     

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.     

Alteration of the disulfide-coupled folding pathway of BPTI by circular permutation

The kinetics of disulfide-coupled folding and unfolding of four circularly permuted forms of bovine pancreatic trypsin inhibitor (BPTI) were studied and compared with previously published results for both wild-type BPTI and a cyclized form. Each of the permuted proteins was found to be less stable than either the wild-type or circular proteins, by 3-8 kcal/mole. These stability differences were used to estimate effective concentrations of the chain termini in the native proteins, which were 1 mM for the wild-type protein and 2.5 to 4000 M for the permuted forms. The circular permutations increased the rates of unfolding and caused a variety of effects on the kinetics of refolding. For two of the proteins, the rates of a direct disulfide-formation pathway were dramatically increased, making this process as fast or faster than the competing disulfide rearrangement mechanism that predominates in the folding of the wild-type protein. These two permutations break the covalent connectivity among the beta-strands of the native protein, and removal of these constraints appears to facilitate direct formation and reduction of nearby disulfides that are buried in the folded structure. The effects on folding kinetics and mechanism do not appear to be correlated with relative contact order, a measure of overall topological complexity. These observations are consistent with the results of other recent experimental and computational studies suggesting that circular permutation may generally influence folding mechanisms by favoring or disfavoring specific interactions that promote alternative pathways, rather than through effects on the overall topology of the native protein.     

Rational design of solution additives for the prevention of protein aggregation

We have developed a statistical-mechanical model of the effect of solution additives on protein association reactions. This model incorporates solvent radial distribution functions obtained from all-atom molecular dynamics simulations of particular proteins into simple models of protein interactions. In this way, the effects of additives can be computed along the entire association/dissociation reaction coordinate. We used the model to test our hypothesis that a class of large solution additives, which we term "neutral crowders," can slow protein association and dissociation by being preferentially excluded from protein-protein encounter complexes, in a manner analogous to osmotic stress. The magnitude of this proposed "gap effect" was probed for two simple model systems: the association of two spheres and the association of two planes. Our results suggest that for a protein of 20 A radius, an 8 A additive can increase the free energy barrier for association and dissociation by as much as 3-6 kcal/mol. Because the proposed gap effect is present only for reactions involving multiple molecules, it can be exploited to develop novel additives that affect protein association reactions although having little or no effect on unimolecular reactions such as protein folding. This idea has many potential applications in areas such as the stabilization of proteins against aggregation during folding and in pharmaceutical formulations.     

Two energetically disparate folding pathways of alpha-lytic protease share a single transition state

The Lysobacter enzymogenes alpha-lytic protease (alphaLP) is synthesized with a 166 amino acid pro region (Pro) that catalyzes the folding of the 198 amino acid protease into its native conformation. An extraordinary feature of this system is the very high energy barrier (DeltaG = 30 kcal mol-1) that effectively prevents alphaLP from folding in the absence of Pro (t1/2 = 1800 years). A pair of mutations has been isolated in the protease that completely suppresses the catalytic defect incurred in Pro by truncation of its last three amino acids. These mutations also accelerate the folding of alphaLP in the absence of Pro by 400-fold. An energetic analysis of the two folding reactions indicates that the mutations stabilize the transition states of both the catalyzed and uncatalyzed folding reactions by 3 kcal mol-1. This finding points to a single transition state for these two distinct and energetically disparate folding pathways, and raises the possibility that all alphaLP folding pathways share the same transition state.     

Effect of proline residues on protein folding

Conformational energy calculations have been used to study the role of the proline residues in the folding of bovine pancreatic trypsin inhibitor. In the calculation, each of the four proline residues of this small protein is forced from the trans to cis peptide isomer while still part of the native folded structure. The cis proline residue can always be accommodated by small changes of the native conformation (< 1 Å root-mean-square deviation). For three of the four proline residues, Pro2, Pro9 and Pro 13, being in the cis form is calculated to destabilize the folded conformation by less than 11 kcal/mol, suggesting that rapid folding to a stable native-like conformation can occur with either isomeric form. For one of these three, Pro13, the destabilization is only 1 kcal/mol, suggesting the existence of an alternative folded native conformation with Pro13 cis. The fourth proline residue, Pro8, is calculated to destabilize the native conformation by so much (33 kcal/mol) that it will block folding in the manner proposed by Brandts et al. (1975).     

Two-dimensional 1H nuclear magnetic resonance study of the (5-55) single-disulphide folding intermediate of bovine pancreatic trypsin inhibitor

An analogue of the bovine pancreatic trypsin inhibitor (BPTI) folding intermediate that contains only the disulphide bond between Cys5 and Cys55 has been prepared in Escherichia coli by protein engineering methods, with the other four Cys residues replaced by Ser. Two-dimensional 1H nuclear magnetic resonance studies of the analogue have resulted in essentially complete resonance assignments of the folded form of the protein. The folded protein has a compact conformation that is structurally very similar to that of native BPTI, although there are subtle differences and the folded conformation is not very stable. Approximately half of the protein molecules are unfolded at 3 degrees C, and this proportion increases at higher temperatures. The folded and unfolded conformations are in slow exchange. The conformational properties of the analogue can explain many aspects of the kinetic role that the normal (5-55) intermediate plays in the folding of BPTI.