Folding Of Bovine Pancreatic Trypsin Inhibitor Research Articles

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.

Divergent folding pathways of two homologous proteins, BPTI and tick anticoagulant peptide: Compartmentalization of folding intermediates and identification of kinetic traps

Bovine pancreatic trypsin inhibitor (BPTI) and tick anticoagulant peptide (TAP) are two structurally homologous proteins, which have been shown to exhibit distinct mechanisms of oxidative folding. We demonstrate here differences of their folding properties using the technique of disulfide scrambling. Two extensively unfolded homologous isomers (beads-form) of BPTI (Cys 5–Cys 14, Cys 30–Cys 38, Cys 51–Cys 55) and TAP (Cys 5–Cys 15, Cys 33–Cys 39, Cys 55–Cys 59) were allowed to refold in parallel via disulfide shuffling of 13 potential isomers to form the native structure. Folding intermediates were trapped by acid quenching and analyzed by HPLC. The results reveal the following diversities: (a) there are two predominant folding intermediates of BPTI and seven well-populated folding intermediates of TAP. One of the two predominant BPTI intermediates (Cys 5–Cys 14, Cys 30 – Cys 51 , Cys 38–Cys 55) contains a native disulfide Cys 30 – Cys 51 and constitutes about 34% of the total BPTI folding intermediates. In contrast, the TAP counterpart (Cys 5–Cys 15, Cys 33 – Cys 55 , Cys 39–Cys 59) represents only 5% of the total TAP intermediates; (b) three isomers of TAP sharing a stable non-native disulfide bond Cys 15–Cys 33 are shown to act as kinetic traps of TAP folding. Their counterparts are conspicuously absent in the BPTI folding; and (c) most significantly, folding intermediates of BPTI are found to be energetically compartmentalized, whereas most folding intermediates of TAP are inter-convertible and exist in dynamic equilibrium. Our studies further demonstrate optimal concentrations of denaturant required for destabilization of kinetic traps and acceleration of TAP folding.

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.

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.

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.

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.

Coarsely resolved topography along protein folding pathways

The kinetic data from the coarse representation of polypeptide torsional dynamics described in the preceding paper [Fernandez and Berry, J. Chem. Phys. 112, 5212 (2000), preceding paper] is inverted by using detailed balance to obtain a topographic description of the potential-energy surface (PES) along the dominant folding pathway of the bovine pancreatic trypsin inhibitor (BPTI). The topography is represented as a sequence of minima and effective saddle points. The dominant folding pathway displays an overall monotonic decrease in energy with a large number of staircaselike steps, a clear signature of a good structure-seeker. The diversity and availability of alternative folding pathways is analyzed in terms of the Shannon entropy σ(t) associated with the time-dependent probability distribution over the kinetic ensemble of contact patterns. Several stages in the folding process are evident. Initially misfolded states form and dismantle revealing no definite pattern in the topography and exhibiting high Shannon entropy. Passage down a sequence of staircase steps then leads to the formation of a nativelike intermediate, for which σ(t) is much lower and fairly constant. Finally, the structure of the intermediate is refined to produce the native state of BPTI. We also examine how different levels of tolerance to mismatches of side chain contacts influence the folding kinetics, the topography of the dominant folding pathway, and the Shannon entropy. This analysis yields upper and lower bounds of the frustration tolerance required for the expeditious and robust folding of BPTI.

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.

Secretion efficiency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability.

Bovine pancreatic trypsin inhibitor (BPTI) has been widely used as a model protein to investigate protein structure and folding pathways. To study the role of its three disulfide bonds in folding, proofreading, and secretion of BPTI in an intact eucaryotic cell, BPTI was expressed and secreted from a synthetic gene in the yeast Saccharomyces cerevisiae. Site-directed mutagenesis was used to create all possible single and pairwise cysteine to alanine BPTI mutants, and the effect of these mutations on secretion efficiency was determined. The 5-55 disulfide bond is found to be essential for secretion-loss of either Cys5, Cys55, or both prevents secretion. Removal of the 14-38 disulfide bond results in a small reduction of secretion, but individual Cys14 or Cys38 replacements reduce secretion efficiency by 30%. Cys30 and Cys30-51 mutants are secreted at half the level of wild-type BPTI, while secretion of the Cys51 mutant is reduced by 90%. BPTI containing only a single disulfide bond (5-55) is not secreted. No relationship is observed between secretion efficiency and in vitro folding or unfolding rates, but mutant BPTI secretion is directly correlated with the in vitro unfolding temperature Tm and the free energy of stabilization provided by each of the three disulfides. These results indicate that structural fluctuations rather than the time-averaged structure observed by NMR or X-ray crystallography may determine recognition of a protein as misfolded and subsequent retention and degradation.

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.

Hydrophobic interactions accelerate early stages of the folding of BPTI.

Bovine pancreatic trypsin inhibitor (BPTI) has long served as an important model system for the studies of the protein folding process. Recently a kinetically important folding intermediate has been detected early on the oxidative folding pathway of BPTI [Dadlez, M., & Kim, P. S. (1995) Nat. Struct. Biol. 2, 674-679]. The intermediate, named [14-38], contains a single native disulfide bond between residues 14 and 38, and forms much faster than any other single-disulfide intermediate. A series of 24 mutants of BPTI has been studied here to detect amino acids which contribute to fast formation of [14-38]. Seven nonpolar or aromatic residues, distant from the cysteines by as many as eight residues, are found to accelerate the formation of 14-38 disulfide, without changing the reactivities of the cysteines. The acceleration is observed even in 8 M urea. It is concluded that in the early stages of the folding of BPTI and BPTI-like domains, the residual structure of the denatured state promotes native pairing of cysteines by way of interaction of hydrophobic residues. A similar mechanism may facilitate early steps in the folding of proteins in general.

A kinetic explanation for the rearrangement pathway of BPTI folding.

Bovine pancreatic trypsin inhibitor (BPTI) does not fold by simple sequential formation of its native disulphide bonds. Instead, an initially formed intermediate, termed N', first rearranges to a more stable species in a slow process that requires substantial unfolding. We find that direct oxidation of N' is also inhibited by native structure which slows both the intermolecular step in oxidation--formation of a mixed disulphide bond with the oxidizing agent GSSG--as well as the subsequent intramolecular step. Folding does not occur appreciably by direct oxidation because the high GSSG concentrations required for efficient mixed disulphide formation cause N' to accumulate as a nonproductive, double-mixed disulphide species. The need to unfold previously acquired native structure, observed in the folding of BPTI, may be a common feature of disulphide-linked folding reactions.

Dynamic structure of a highly ordered beta-sheet molten globule: multiple conformations with a stable core.

The structure of [14-38]Abu, a variant of bovine pancreatic trypsin inhibitor (BPTI) with only the 14-38 disulfide bridge intact, has been analyzed by two-dimensional 1H and 1H-15N NMR. Except for the 18-24, 29-35 antiparallel beta-sheet, residues in all regions of the molecule give two exchange cross peaks for each 1H; for one residue, Gly 37, three exchange cross peaks are observed. The presence of exchange cross peaks indicates that the residues sample conformations that interconvert on a time scale of milliseconds or longer. Over 90% of the NMR spectra have been assigned, including backbone and side chain atoms and their exchange cross peaks. Analyses of chemical shifts, chemical exchange, hydrogen isotope exchange, and NOEs indicate that [14-38]Abu at pH 4.5 and 1 degree C is an ensemble of interconverting conformations, in all of which the 18-24, 29-35 antiparallel beta-sheet is native-like and intact. Outside the antiparallel beta-sheet, residues undergo local order/disorder transitions. The stable structure of [14-38]Abu is not in the vicinity of the 14-38 disulfide bond but rather is in the slow-exchange core. NOE analysis indicates that the main tertiary interactions involve hydrophobic contacts with the rings of Tyr 21, Tyr 23, and Tyr 35. As a model for early folding intermediates, the structure of [14-38]Abu suggests that BPTI folding is initiated by stabilization of a turn existing in the unfolded protein and involves both local and nonlocal hydrophobic interactions.

A third native one-disulphide intermediate in the folding of bovine pancreatic trypsin inhibitor.

A previously unidentified intermediate has been detected in the early stages of the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI). The intermediate contains one disulphide bond between residues 14 and 38 and is denoted [14-38]. The 14-38 disulphide bond is also found in native BPTI. Although the other native one-disulphide intermediates, [30-51] and [5-55], are thermodynamically more stable, [14-38] can be populated substantially at the early stages of BPTI folding. Moreover, initial characterization of the kinetic properties of this intermediate strongly suggest that a substantial fraction of BPTI molecules fold by way of the [14-38] intermediate. Our results emphasize the importance of native-like tendencies in protein folding.

The stabilizing effects of hydrophobic cores on peptide folding of bovine-pancreatic-trypsin-inhibitor folding-intermediate model.

A synthetic peptide model composed of alpha-helical and beta-sheet portions (P alpha P beta) is a crucial folding intermediate in the folding of bovine pancreatic trypsin inhibitor (BPTI), which contains 30 amino acid residues, and provides a good model for studying the folding structure. Using this model the roles of hydrophobic cores, such as non-polar amino acids and a short beta strand, in the folding structure stability were investigated by deleting the hydrophobic amino acids or substituting with Ala. As a first step, a mutant peptide, P alpha 6 P beta 1, was made by removing the four residues (NNFK46) from the N-terminus of P alpha which make a short beta strand in native BPTI, and each of the hydrophobic cores, Phe45 of P alpha and Tyr21 of P beta, were substituted by Ala. Without the short beta strand the peptide still folds in spite of its low solubility, suggesting that a simple alpha helix and central antiparallel beta sheet contain sufficient information to direct the folding of this region of molecule. The peptide without Tyr21 was much more unstable than P alpha 6 P beta 1, such that most of the folding structure collapsed even at 0 degree C, whereas without Phe45 the structure was more stable than P alpha 6 P beta 1. This indicates that the hydrophobic interactions of Tyr21 in P beta are more important in BPTI folding.

Effects of negative charges of a model for bovine pancreatic trypsin inhibitor folding intermediate on the peptide folding.

A peptide model (called P alpha P beta) of bovine pancreatic trypsin inhibitor (BPTI) for studying the peptide folding structure was designed as a crucial folding intermediate. The P alpha P beta model folded but it was unstable at low pH. Four acidic groups in P alpha P beta: Glu-49, Asp-50, and C-termini at Phe-33 and Ala-58, were replaced individually with an Ala (in case of Glu and Asp) and an amide group (for C-terminal carboxyl groups) to study the effects of these negative charged groups on folding structure in terms of thermal stability and pH dependence. Substituting the Glu-49 or Asp-50 with Ala and blocking the C-terminus carboxyl group of Phe-33 in P beta destabilized the structure, but blocking the negative charge of C-terminus in Ala-58 of P alpha stabilized the structure at neutral pH. These results can be interpreted in terms of helix dipole momentum effects and/or salt bridge formation between Asp-50 and Arg-53, and of electrostatic interaction between positive and negative charges, which stabilized the structure. The melting temperature of model peptides (Tm) in low ionic strength buffer were lower than that in high ionic strength buffer except the C-terminus blocking of P alpha.

Efficient catalysis of disulphide bond rearrangements by protein disulphide isomerase.

Protein disulphide isomerase (PDI) is a highly abundant and ubiquitous eukaryotic protein that is essential for viability in yeast. Although PDI is thought to catalyse disulphide bond formation and isomerization during protein biosynthesis, PDI has been found previously to have only moderate effects (approximately 25-fold) on the rate of oxidative folding of proteins in vitro. In addition, PDI has been implicated in several apparently unrelated cellular functions. For example, PDI is the beta-subunit of prolyl 4-hydroxylase and is part of the triglyceride transfer complex. The oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) is slow and inefficient in vitro. Here we report that PDI increases by a factor of 3,000-6,000 the rates of folding of kinetically trapped BPTI folding intermediates, in which native structure impedes disulphide bond formation. By contrast, PDI has only small effects on the rate of disulphide bond formation in intermediates that are oxidized readily in the absence of PDI. These results suggest that an important function of PDI is to catalyse disulphide bond formation and rearrangements within kinetically trapped, structured folding intermediates.

Native and non-native intermediates in the BPTI folding pathway.

Recent studies of the disulfide-bonded intermediates in the refolding of bovine pancreatic trypsin inhibitor (BPTI) indicate that the most stable intermediates can take on much or all of the structure of the fully folded protein. Native-like structure in the intermediates probably causes steric inhibition of direct sequential formation of the three disulfides found in the native protein, thus accounting for the role of intramolecular rearrangements in the folding mechanism of this small protein.

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 °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.

Reexamination of the Folding of BPTI: Predominance of Native Intermediates

Bovine pancreatic trypsin inhibitor (BPTI) continues to be the only protein for which a detailed pathway of folding has been described. Previous studies led to the conclusion that nonnative states are well populated in the oxidative folding of BPTI. This conclusion has broadly influenced efforts to understand protein folding. The population of intermediates present during the folding of BPTI has been reexamined by modern separation techniques. It was found that all well-populated folding intermediates contain only native disulfide bonds. These data emphasize the importance of native protein structure for understanding protein folding.