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.     

Kinetic roles and conformational properties of the non-native two-disulphide intermediates in the refolding of bovine pancreatic trypsin inhibitor.

The most productive folding pathway of reduced bovine pancreatic trypsin inhibitor (BPTI) proceeds through the disulphide intermediates (30-51), (30-51, 5-14), and (30-51, 5-38); these are important kinetic intermediates in folding, even though the latter pair contain non-native disulphide bonds. Analogues of these intermediates have been prepared by protein engineering methods and their conformational properties examined by circular dichroism and 1H-nuclear magnetic resonance. The (30-51), (30-51, 5-14) and (30-51, 5-38) analogues exhibit comparable degrees of stable structure, which cannot include those portions of the polypeptide chain involving Cys5, Cys14 and Cys38. These properties are consistent with the roles of (30-51, 5-14) and (30-51, 5-38) in the folding pathway of BPTI, which demand that they exhibit a considerable degree of conformational flexibility in part of the molecule.     

Partially folded bovine pancreatic trypsin inhibitor analogues attain fully native structures when co-crystallized with S195A rat trypsin

Crystal structures, at 1.7 Å resolution, were solved for complexes between each of two chemically synthesized partially folded analogues of bovine pancreatic trypsin inhibitor (BPTI) with the proteolytically inactive rat trypsin mutant S195A. The BPTI analogue termed [14-38]_Abu retains only the disulfide bond between Cys14 and Cys38, while Cys5, Cys30, Cys51, and Cys55 are replaced by isosteric α-amino-n-butyric acid residues. The analogue K26P,A27D[14-38]_Abu contains two further replacements, by statistically favored residues, in the type I β-turn that has been suggested to be a main site for initiation of BPTI folding. As a control, the structure of the complex between S195A trypsin and wild-type BPTI was also solved. Despite significant differences in the degree of structure detected among these three BPTIs in solution by several biophysical techniques, their tertiary folds once bound to S195A trypsin in a crystalline lattice are essentially superimposable.     

Phi-values for BPTI folding intermediates and implications for transition state analysis

Amino acid replacements were used to probe the roles of 14 sites in two well-characterized intermediates in the folding pathway of bovine pancreatic trypsin inhibitor (BPTI). One of these intermediates contains one of the three disulfides found in the native protein (30--51). NMR studies have shown that approximately two-thirds of this polypeptide has a native-like conformation. The other intermediate contains two native disulfides (30--51 and 5--55) and has a fully folded conformation. The phi-values for a majority of residues were <1, indicating that the native protein was significantly more destabilized than either intermediate even when the altered residue was located in a well-ordered region of the intermediate. These observations suggest that folding intermediates and transition states may generally be more structured than indicated by phi-values alone.     

Formation of a native-like subdomain in a partially folded intermediate of bovine pancreatic trypsin inhibitor.

In the folding of bovine pancreatic trypsin inhibitor (BPTI), the single-disulfide intermediate [30-51] plays a key role. We have investigated a recombinant analog of [30-51] using a 2-dimensional nuclear magnetic resonance (2D-NMR). This recombinant analog, named [30-51]Ala, contains a disulfide bond between Cys-30 and Cys-51, but contains alanine in place of the other cysteines in BPTI to prevent the formation of other intermediates. By 2D-NMR, [30-51]Ala consists of 2 regions-one folded and one predominantly unfolded. The folded region resembles a previously characterized peptide model of [30-51], named P alpha P beta, that contains a native-like subdomain with tertiary packing. The unfolded region includes the first 14 N-terminal residues of [30-51] and is as unfolded as an isolated peptide containing these residues. Using protein dissection, we demonstrate that the folded and unfolded regions of [30-51]Ala are structurally independent. The partially folded structure of [30-51]Ala explains many of the properties of authentic [30-51] in the folding pathway of BPTI. Moreover, direct structural characterization of [30-51]Ala has revealed that a crucial step in the folding pathway of BPTI coincides with the formation of a native-like subdomain, supporting models for protein folding that emphasize the formation of cooperatively folded subdomains.     

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.     

Hydrogen exchange in BPTI variants that do not share a common disulfide bond.

Bovine pancreatic trypsin inhibitor (BPTI) is stabilized by 3 disulfide bonds, between cysteines 30-51, 5-55, and 14-38. To better understand the influence of disulfide bonds on local protein structure and dynamics, we have measured amide proton exchange rates in 2 folded variants of BPTI, [5-55]Ala and [30-51; 14-38]V5A55, which share no common disulfide bonds. These proteins resemble disulfide-bonded intermediates that accumulate in the BPTI folding pathway. Essentially the same amide hydrogens are protected from exchange in both of the BPTI variants studied here as in native BPTI, demonstrating that the variants adopt fully folded, native-like structures in solution. However, the most highly protected amide protons in each variant differ, and are contained within the sequences of previously studied peptide models of related BPTI folding intermediates containing either the 5-55 or the 30-51 disulfide bond.     

Early events in the disulfide-coupled folding of BPTI.

Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14-38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide-formation steps were carried out. The results of these studies indicate that the formation of [14-38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 degrees C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of [14-38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.     

The structure of denatured bovine pancreatic trypsin inhibitor (BPTI)

In the presence of denaturant and thiol initiator, the native bovine pancreatic trypsin inhibitor (BPTI) denatures by shuffling its native disulfide bonds and converts to a mixture of scrambled isomers. The extent of denaturation is evaluated by the relative yields of the scrambled and native species of BPTI. BPTI is an exceedingly stable molecule and can be effectively denatured only by guanidine thiocyanate (GdmSCN) at concentrations higher than 3-4 M. The denatured BPTI consists of at least eight fractions of scrambled isomers. Their composition varies under increasing concentrations of GdmSCN. In the presence of 6 M GdmSCN, the most predominant fraction of scrambled BPTI accounts for 56% of the total structure of denatured BPTI. Structural analysis reveals that this predominant fraction contains the bead-form isomer of scrambled BPTI, bridged by three pairs of neighboring cysteines, Cys5-Cys14, Cys30-Cys38 and Cys51-Cys55. The extreme conformational stability of BPTI has important implications in its distinctive folding pathway.     

Native-like interactions favored in the unfolded bovine pancreatic trypsin inhibitor have different roles in folding

Folding kinetics of a series of bovine pancreatic trypsin inhibitor (BPTI) variants with similar stabilities and structures have been measured. All are strongly destabilized relative to WT. In Y21A, F22A, Y23A, G37A, and F45A, the three native disulfide bonds are retained. In RM(14-38), Cys14 and Cys38 thiols are methylated while C30-C51 and C5-C55 disulfides remain intact. At pH 2 and 20 degrees C, relaxation rate constants of the major kinetic phase range from approximately 10 ms to 0.71 s in the absence of denaturant. All mutants except G37A exhibit standard two-state behavior. Y21A, F22A, and Y23A fold much more slowly than other mutants. The experiments were designed to test the hypothesis that native-like structure detected in the unfolded BPTI is important in folding. Two native-like contacts are implied by NOEs in reduced and unfolded BPTI, between residues Tyr23 and Ala25, and between Gly37 NH and the Tyr35 ring. The results support an earlier hypothesis that formation of the central beta-hairpin, monitored by a local native interaction between Tyr23 and Ala25, is crucial to initiation of BPTI folding. The second native-like contact is important, not in folding initiation, but in preventing a kinetic trap later in the process. Evidence for this comes from mutant G37A, which behaves very differently from the others in displaying a phenomenon called rollover. G37A is, to our knowledge, the first reported case in which a single-site replacement causes rollover, while the wild type and all other known mutants of the same protein show typical two-state chevron plots. The best explanation is that the G37A mutation introduces a kinetic trap of the type described by Chan and Dill [(1998) Proteins 30, 2-33]. In native BPTI, there is an unusual polar interaction between the ring of Tyr35 and the backbone NH of Gly37. Our results suggest that the NH-aromatic interaction between residues 37 and 35 is important throughout folding in stabilizing native-like loop conformations and in preventing the flexible loops from being trapped in nonfunctional conformations during later stages of folding.     

Engineering an unnatural Nalpha-anchored disulfide into BPTI by total chemical synthesis: structural and functional consequences

A disulfide-engineered analogue of bovine pancreatic trypsin inhibitor (BPTI), ((N(alpha)-(CH2)2S-)Gly38)BPTI, has been prepared using a thioester-mediated auxiliary functional group chemical ligation of a N(alpha)-ethanethiol-containing peptide segment with a peptide-alphaCOSR segment. In this study, Nalpha-(ethanethiol)Gly38 replaces the native Cys38, providing the sulfhydryl group required for ligation and folding. Comparisons between ((Nalpha-(CH2)2SH)Gly38)BPTI, synthetic native BPTI and reference BPTI purchased from Sigma were made using mass spectroscopy, enzyme inhibitor association constant determination (K(a)) and 1H-nuclear magnetic resonance total correlated spectroscopy (1H-NMR TOCSY) measurements. The K(a) value for ((Nalpha-(CH2)2SH)Gly38)BPTI was approximately 20-fold lower than synthetic and reference BPTI, which was attributed to perturbations in the binding loop of the protein (near Cys14). This hypothesis was confirmed by two-dimensional (2D) 1H-NMR TOCSY experiments. The data reported here demonstrate that total chemical synthesis by auxiliary functional group chemical ligation is a practical method for the synthesis of a novel class of biologically active protein analogues containing additional functional groups linked to the protein backbone.     

Conformational restrictions on the pathway of folding and unfolding of the pancreatic trypsin inhibitor

The kinetic roles of the partially folded, intermediate protein species with two disulphide bonds in folding and unfolding of the pancreatic trypsin inhibitor have been investigated further. Formation of a second disulphide bond between Cys5 and Cys55 during refolding of the reduced inhibitor, which would yield the species with the 30–51 and 5–55 disulphide bonds and, possibly, the native-like conformation of the protein, is not significant. Instead, three other second disulphide bonds (5–14, 5–38 and 14–38) are formed approximately 10⁵ times more readily, but each of these two-disulphide species then rearranges intramolecularly to the native-like, two-disulphide intermediate. Therefore, the reduced protein does not simply form sequentially the three disulphide bonds of the native state. Unfolding of the native state takes place by the reverse of this process.The kinetic importance for folding and unfolding of this transition between two-disulphide intermediates under the conditions used here was illustrated experimentally by a modified form of the inhibitor in which the thiols of Cys14 and Cys38 were blocked irreversibly. In the folded conformation, this modified protein is more stable to unfolding than normal, but after unfolding cannot readily regain the native-like conformation, because Cys14 or Cys38 are required to be involved in disulphide bonds during the interconversion of the two-disulphide intermediates.Some conception of the conformational transitions that take place at each stage of the folding transition may be inferred from the relative propensities of the six cysteine residues to make or rearrange disulphide bonds. It is concluded that the inhibitor probably does not refold by sequential adoption of the native conformation by the unfolded polypeptide chain. Instead, it appears that essentially all elements of the native conformation are attained simultaneously in the final stage of folding, within an unstable and flexible, yet relatively compact, form of the entire polypeptide chain produced by weak interactions between groups distant in the primary structure.     

Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds.

The equilibrium and kinetic behavior of the guanidine hydrochloride (Gdn-HCl) induced unfolding/refolding of four bovine pancreatic trypsin inhibitor (BPTI) mutants was examined by using ultraviolet difference spectroscopy. In three of the mutants, we replaced the buried 30-51 disulfide bond with alanine at position 51 and valine (Val30/Ala51), alanine (Ala30/Ala51), or threonine (Thr30/Ala51) at position 30. For the fourth mutant, the solvent-exposed 14-38 disulfide was substituted by a pair of alanines (Ala14/Ala38). All mutants retained the 5-55 disulfide. Experiments were performed under oxidizing conditions; thus, both the unfolded and folded forms retained two native disulfide bonds. Equilibrium experiments demonstrated that all four mutants were destabilized relative to wild-type BPTI. However, the stability of the 30-51 mutants increased with the hydrophobicity of the residue substituted at position 30. Kinetic experiments showed that all four mutants contained two minor slow refolding phases with characteristics of proline isomerization. The specific behavior of the phases depended on the location of the disulfide bonds. The major unfolding/refolding phase for each of the 30-51 mutants was more than an order of magnitude slower than for Ala14/Ala38 or for BPTI in which the 14-38 disulfide bond was specifically reduced and blocked with iodoacetamide [Jullien, M., & Baldwin, R. L. (1981) J. Mol. Biol. 145, 265-280]. Since this effect is independent of the stability of the protein, it is consistent with a model in which the proper docking of the interior residues of the protein is the rate-limiting step in the folding of these mutants.     

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.     

The 5-55 single-disulphide intermediate in folding of bovine pancreatic trypsin inhibitor.

An analogue of the BPT1 folding intermediate that contains only the disulphide bond between Cys-5 and Cys-55 has been prepared by mutation of the other four Cys residues to Ser. On the basis of its circular dichroism and 1H-nuclear magnetic resonance spectra and its electrophoretic mobility, this intermediate is shown to be at least partially folded at low temperatures. This probably accounts for several of the unique properties of this intermediate observed during folding.     

A major kinetic trap for the oxidative folding of human epidermal growth factor

The folding pathway of human epidermal growth factor (EGF) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. Oxidative folding of the fully reduced EGF proceeds through 1-disulfide intermediates and accumulates rapidly as a single stable 2-disulfide intermediate (designated as EGF-II), which represents up to more than 85% of the total protein along the folding pathway. Among the five 1-disulfide intermediates that have been structurally characterized, only one is native, and nearly all of them are bridges by neighboring cysteines. Extensive accumulation of EGF-II indicates that it accounts for the major kinetic trap of EGF folding. EGF-II contains two of the three native disulfide bonds of EGF, Cys(14)-Cys(31) and Cys(33)-Cys(42). However, formation of the third native disulfide (Cys(6)-Cys(20)) for EGF-II is slow and does not occur directly. Kinetic analysis reveals that an important route for EGF-II to reach the native structure is via rearrangement pathway through 3-disulfide scrambled isomers. The pathway of EGF-II to attain the native structure differs from that of three major 2-disulfide intermediates of bovine pancreatic trypsin inhibitor (BPTI). The dissimilarities of folding mechanism(s) between EGF, BPTI, and hirudin are discussed in this paper.     

A new two-disulphide intermediate in the refolding of reduced bovine pancreatic trypsin inhibitor

The apparently complete refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) is shown to produce a mixture of two species. One of these is native BPTI, but the other lacks the disulphide bond between cysteines 30 and 51. The latter species has a folded conformation very like that of native BPTI, and is oxidized by air to native BPTI on warming in aqueous solution. The two unreactive cysteine thiol groups appear to be buried in the interior of the molecule, which restricts access by reagents that can alkylate them or oxidize them to form the disulphide bond. The implications of this intermediate and its conformation for the understanding of protein folding are discussed.     

Solid-phase synthesis of bovine pancreatic trypsin inhibitor (BPTI) and two analogues. A chemical approach for evaluating the role of disulfide bridges in protein folding and stability.

The linear sequence of bovine pancreatic trypsin inhibitor (BPTI) has been assembled by stepwise Fmoc solid-phase peptide synthesis on a polyethylene glycol-polystyrene (PEG-PS) graft support with p-alkoxybenzyl ester anchoring. Similar methods were used to prepare two analogues, the first with all six half-cystine (Cys) residues replaced by alpha-amino-n-butyric acid (Abu), and the second with replacement of Abu at four Cys positions while retaining the native pairing between positions 14 and 38. Following cleavage from the support, the linear molecules (reduced form) were purified by semipreparative reversed-phase high performance liquid chromatography (HPLC). The native structure of BPTI was then formed by oxidation of a dilute solution of the protein at pH 8.7 in the presence of oxidized glutathione. The BPTI analogue with one disulfide bridge was obtained following treatment with dimethyl sulfoxide (DMSO)-pH 6 buffer (1:9). Overall yields of homogeneous proteins were 2-4%, and further characterization was provided by amino acid analysis, sequencing, ion electrospray mass spectrometry, analytical HPLC, and capillary zone electrophoresis (CZE). Purified synthetic BPTI with the native sequence was indistinguishable from natural material by the analytical and biophysical criteria applied, including circular dichroism (CD) spectra and inhibition of trypsin action. Studies are in progress to evaluate conformational features of the analogues which respectively lack two, or all three, of the native disulfide bridges.     

The single-disulphide intermediates in the refolding of reduced pancreatic trypsin inhibitor

The intermediate species with one disulphide bond in the renaturation of reduced pancreatic trypsin inhibitor have been trapped, isolated, and the Cys residues involved in the disulphide bonds determined. Approximately half the intermediate species had the disulphide bond between Cys-30 and 51, a disulphide bond also present in the native inhibitor. The next most predominant species, representing one-quarter of the total, had a disulphide bond between Cys-5 and 30, and two more minor species involving Cys-30 and 55 and Cys-5 and 51 were detected; these disulphide bonds are not present in the native inhibitor.The nature of the disulphide bonds present are concluded to reflect primarily the conformational forces acting at this stage of folding, which may be primarily interactions between segments with propensities for secondary structure, either helices or β-sheet. The general importance of such interactions in protein folding is discussed.     

Structural stability of disulfide mutants of basic pancreatic trypsin inhibitor: A molecular dynamics study

The structure and folding of basic pancreatic trypsin inhibitor (BPTI) has been studied extensively by experimental means. We report a computer simulation study of the structural stability of various disulfide mutants of BPTI, involving eight 250-psec molecular dynamics simulations of the proteins in water, with and without a phosphate counterion. The presence of the latter alters the relative stability of the single disulfide species [5–55] and [30–51]. This conclusion can explain results of mutational studies and the conservation of residues in homologues of BPTI, and suggests a possible role of ions in stabilizing one intermediate over another in unfolding or folding processes. © 1996 Wiley-Liss, Inc.