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.     

The underlying mechanism for the diversity of disulfide folding pathways

The disulfide folding pathway of bovine pancreatic trypsin inhibitor (BPTI) is characterized by the predominance of folding intermediates with native-like structures. Our laboratory has recently analyzed the folding pathway(s) of four 3-disulfide-containing proteins, including hirudin, potato carboxypeptidase inhibitor, epidermal growth factor, and tick anticoagulant peptide. Their folding mechanism(s) differ from that of BPTI by 1) a higher degree of heterogeneity of 1- and 2-disulfide intermediates and 2) the presence of 3-disulfide scrambled isomers as folding intermediates. To search for the underlying causes of these diversities, we conducted kinetic analyses of the reductive unfolding of these five proteins. The experiment of reductive unfolding was designed to evaluate the relative stability and interdependence of disulfide bonds in the native protein. It is demonstrated here that among these five proteins, there exists a striking correlation between the mechanism(s) of reductive unfolding and that of oxidative folding. Those proteins with their native disulfide bonds reduced in a collective and simultaneous manner exhibit both a high degree of heterogeneity of folding intermediates and the accumulation of scrambled isomers along the folding pathway. A sequential reduction of the native disulfide bonds is associated with the presence of predominant intermediates with native- like structures.     

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.     

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.     

Mechanism of protein folding. IV. Forming and breaking of disulfide bonds in bovine pancreatic tripsin inhibitor.

The folding mechanism of bovine pancreatic tripsin inhibitor (BPTI) is explained theoretically on the basis of the island model, where the driving force of folding is hydrophobic interaction. For this purpose, we take a look at the formation and breaking of disulfide bonds during the folding process of BPTI. The intermediate conformations and the native one are successfully obtained, which satisfy the so-called "lampshade" geometrical criterion for the formation of the disulfide bonds. The folding pathway is consistent with the renaturation experiment by Creighton. In addition, an elaborate treatment of side chains of amino acid residues by the software programme CHARMm confirms quantitatively the formation of disulfide bridges.     

Evidence for the underlying cause of diversity of the disulfide folding pathway

The pathways of oxidative folding of disulfide proteins exhibit a high degree of diversity, which is illustrated by the varied extent of (a) the heterogeneity of folding intermediates, (b) the predominance of intermediates containing native disulfide bonds, and (c) the level of accumulation of fully oxidized scrambled isomers as intermediates. BPTI and hirudin exemplify two extreme cases of such divergent folding pathways. We previously proposed that the underlying cause of this diversity is associated with the degree of stability of protein subdomains. Here we present compelling evidence that substantiates this hypothesis by studying the folding pathway of alphaLA-IIA. alphaLA-IIA is a partially folded intermediate of alpha-lactalbumin (alphaLA). It comprises a structured beta-sheet (calcium-binding) domain linked by two native disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) and a disordered alpha-helical domain with four free cysteines (Cys(6), Cys(28), Cys(111), and Cys(120)). Purified alphaLA-IIA was allowed to refold without and with stabilization of its structured beta-sheet domain by calcium. In the absence of calcium, the folding pathway of alphaLA-IIA resembles that of hirudin, displaying a highly heterogeneous population of folding intermediates, including fully oxidized scrambled species. Upon stabilization of its beta-sheet domain by bound calcium, oxidative folding of alphaLA-IIA undergoes a pathway conspicuously similar to that of BPTI, exhibiting limited species of folding intermediates containing mostly native disulfide bonds.     

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.     

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.     

The pro region of BPTI facilitates folding.

The in vitro folding pathway of bovine pancreatic trypsin inhibitor (BPTI) has been described previously in terms of the disulfide-bonded intermediates that accumulate during folding of the protein. Folding is slow, occurring in hours at pH 7.3, 25 degrees C. In addition, approximately half of the BPTI molecules become trapped as a dead-end, native-like intermediate. In vivo, BPTI is synthesized as a precursor protein that includes a 13 residue amino-terminal pro region. This pro region contains a cysteine residue. We find that, in vitro, both the rate of formation and the yield of properly folded BPTI are increased substantially in a recombinant model of pro-BPTI. The cysteine residue is necessary for this effect. Moreover, a single cysteine residue, tethered to the carboxy-terminal end of BPTI with a flexible linker of repeating Ser-Gly-Gly residues, is sufficient to assist in disulfide formation. Thus, the pro region appears to facilitate folding by providing a tethered, solvent-accessible, intramolecular thiol-disulfide reagent.     

Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A.

The X-ray structure of a variant of basic pancreatic trypsin inhibitor (BPTI) has been analyzed to determine the structural accommodation resulting from removal of a disulfide cross-link in a protein. The disulfide removed, Cys30-Cys51, has been implicated in both the folding pathway of the protein and its overall thermal stability. In the variant studied, C30A/C51A, the disulfide cysteines were replaced by less bulky alanines. The atomic displacements observed for C30A/C51A indicate a set of concerted shifts of two segments of chains, which together significantly diminish a packing defect at the site of the removed cysteine sulfur atoms. The observed structural changes are distributed asymmetrically around the sites of mutation, indicating that the adjacent beta-sheet is more resistant to the perturbation than the alpha-helix on the opposite side of the disulfide bond. The thermal parameters of groups involved in the structural accommodation are not significantly altered. A comparison of the X-ray structures reported for native BPTI determined in three different crystal forms indicates that the magnitude of its conformational variability exceeds that of the structural changes caused by the disulfide removal. This emphasizes the necessity of using isomorphous crystal systems to determine the relatively small effects due to mutation.     

Differential cooperative enzymatic activities of protein disulfide isomerase family in protein folding

Endoplasmic reticulum (ER)p61, ERp72, and protein disulfide isomerase (PDI), which are members of the PDI family protein, are ubiquitously present in mammalian cells and are thought to participate in disulfide bond formation and isomerization. However, why the 3 different members need to be colocalized in the ER remains an enigma. We hypothesized that each PDI family protein might have different modes of enzymatic activity in disulfide bond formation and isomerization. We purified PDI, ERp61, and ERp72 proteins from rat liver microsomes and compared the effects of each protein on the folding of bovine pancreatic trypsin inhibitor (BPTI). ERp61 and ERp72 accelerated the initial steps more efficiently than did PDI. ERp61 and ERp72, however, accelerated the rate-limiting step less efficiently than did PDI. PDI or ERp72 did not impede the folding of BPTI by each other but rather catalyzed the folding reaction cooperatively with each other. These data suggest that differential enzymatic activities of ERp proteins and PDI represent a complementary contribution of these enzymes to protein folding in the ER.     

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.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

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].     

Selenoglutathione: efficient oxidative protein folding by a diselenide

Diselenide bonds are intrinsically more stable than disulfide bonds. To examine how this stability difference affects reactivity, we synthesized selenoglutathione (GSeSeG), an analogue of the oxidized form of the tripeptide glutathione that contains a diselenide bond in place of the natural disulfide. The reduction potential of this diselenide bond was determined to be -407 +/- 9 mV, a value which is 151 mV lower than that of the disulfide bond in glutathione (GSSG). Thus, the diselenide bond of GSeSeG is 7 kcal/mol more stable than the disulfide bond of GSSG. Nonetheless, we found that GSeSeG can be used to oxidize cysteine residues in unfolded proteins, a process that is driven by the gain in protein conformational stability upon folding. Indeed, the folding of both ribonuclease A (RNase A) and bovine pancreatic trypsin inhibitor (BPTI) proceeded efficiently using GSeSeG as an oxidant, in the former case with a 2-fold rate increase relative to GSSG and in the latter case accelerating conversion of a stable folding intermediate to the native state. In addition, GSeSeG can also oxidize the common biological cofactor NADPH and is a good substrate for the NADPH-dependent enzyme glutathione reductase (kcat = 69 +/- 2 s-1, Km = 54 +/- 7 microM), suggesting that diselenides can efficiently interact with the cellular redox machinery. Surprisingly, the greater thermodynamic stability of diselenide bonds relative to disulfide bonds is not matched by a corresponding decrease in reactivity.     

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.     

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.     

Mutational analysis of the BPTI folding pathway: I. Effects of aromatic-->leucine substitutions on the distribution of folding intermediates.

The roles of aromatic residues in determining the folding pathway of bovine pancreatic trypsin inhibitor (BPTI) were analyzed mutationally by examining the distribution of disulfide-bonded intermediates that accumulated during the refolding of protein variants in which tyrosine or phenylalanine residues were individually replaced with leucine. The eight substitutions examined all caused significant changes in the intermediate distribution. In some cases, the major effect was to decrease the accumulation of intermediates containing two of the three disulfides found in the native protein, without affecting the distribution of earlier intermediates. Other substitutions, however, led to much more random distributions of the intermediates containing only one disulfide. These results indicate that the individual residues making up the hydrophobic core of the native protein make clearly distinguishable contributions to conformation and stability early in folding: The early distribution of intermediates does not appear to be determined by a general hydrophobic collapse. The effects of the substitutions were generally consistent with the structures of the major intermediates determined by NMR studies of analogs, confirming that the distribution of disulfide-bonded species is determined by stabilizing interactions within the ordered regions of the intermediates. The plasticity of the BPTI folding pathway implied by these results can be described using conformational funnels to illustrate the degree to which conformational entropy is lost at different stages in the folding of the wild-type and mutant proteins.     

Pathway of Oxidative Folding of a 3-Disulfide α-Lactalbumin May Resemble Either BPTI Model or Hirudin Model

Pathways of oxidative folding of disulfide proteins display a high degree of diversity and vary among two extreme models. The BPTI model is defined by limited species of folding intermediates adopting mainly native disulfide bonds. The hirudin model is characterized by highly heterogeneous folding intermediates containing mostly non-native disulfide bonds. αLA-IIIA is a 3-disulfide variant of α-lactalbumin (αLA) with a 3-D conformation essentially identical to that of intact αLA. αLA-IIIA contains 3 native disulfide bonds of αLA, two of them are located at the calcium binding β-subdomain (Cys⁶¹–Cys⁷⁷ and Cys⁷³–Cys⁹¹) and the third bridge is located within the α-helical domain of the molecule (Cys²⁸–Cys¹¹¹). We investigate here the pathway of oxidative folding of fully reduced αLA-IIIA with and without stabilization of its β-subdomain by calcium binding. In the absence of calcium, the folding pathway of αLA-IIIA was shown to resemble that of hirudin model. Upon stabilization of β-sheet domain by calcium binding, the folding pathway of αLA-IIIA exhibits a striking similarity to that of BPTI model. Three predominant folding intermediates of αLA-IIIA containing exclusively native disulfide bonds were isolated and structurally characterized. Our results further demonstrate that stabilization of subdomains in a protein may dictate its folding pathway and represent a major cause for the existing diversity in the folding pathways of the disulfide-containing proteins.     

Confirmation of the predicted source of a slow folding reaction: proline 8 of bovine pancreatic trypsin inhibitor

A major question in protein structural analysis concerns the applicability of results from model systems to other proteins. Theoretical approaches seem the best manner of transferring information from one system to another, but their accuracy in the model systems must first be tested with results from experiment. Since bovine pancreatic trypsin inhibitor (BPTI) is a model system for the evaluation of energy minimization and molecular dynamics routines, we can use folding and stability measurements to examine the reliability of these methods. All two-disulfide mutants of BPTI investigated thus far have two very slow folding reactions which have characteristics of proline isomerization. These reactions may occur because the non-native cis form of two of the four prolines in BPTI significantly destabilizes the folded state of the protein. Previous energy minimization studies of wild-type BPTI suggested that the cis form of Pro8 was the most destabilizing of the four prolines [Levitt,M. (1981) J. Mol. Biol., 145, 251-263]. In this paper, we show that mutation of Pro8----Gln in the two-disulfide bond mutant Val30/Ala51 results in a loss of the slowest folding reaction, consistent with Levitt's prediction.