Bovine Pancreatic Trypsin Inhibitor Variants Research Articles

Structure of single-disulfide variants of bovine pancreatic trypsin inhibitor (BPTI) as probed by their binding to bovine β-trypsin

Native bovine pancreatic trypsin inhibitor (BPTI) contains three disulfide bonds: Cys5–Cys55, Cys14–Cys38 and Cys30–Cys51. Correct cysteine pairing, native structure, and full anti-proteinase activity can be restored in the process of oxidative refolding of reduced BPTI. Oxidative refolding starts with the formation of single disulfide intermediates. All 15 single-disulfide variants of BPTI (three native and 12 non-native combinations) have been expressed in Escherichia coli . In each variant the remaining four cysteine residues were replaced by alanine. Four of these variants are shown here to inhibit bovine β-trypsin: three of them contain native and one non-native (Cys5–Cys51) disulfide. All but one (Cys5–Cys55) variant are slowly digested by the enzyme, therefore measurements were performed at pH 4.0, at which trypsin activity is low. Binding constants of these four single disulfide variants were at least two orders of magnitude lower than for native BPTI. Remarkably, in some of the variants the bonding constants were found to be higher for the reduced rather than for the oxidized form of the variant. Also for the fully reduced native BPTI, determined here, the binding constant is of considerable value. Two sets of control experiments demonstrated that the binding of reduced native BPTI to trypsin is specific. In the first, mutation of Lys15 (P1 position) in the binding loop abolished binding of the reduced forms to trypsin. In the second, the binding of reduced native BPTI to anhydrotrypsin yielded the expected UV difference spectra. In general, the results obtained indicate that the inhibitor activity can be induced even in the reduced protein. This activity is not a local effect, such as the nature of residues surrounding the binding loop, but rather is induced by residual structure in the unfolded protein. This structure has been shown to consist of a set of hydrophobic residues and the data presented here indicate that reduced cysteine residues provide further stabilization of such a hydrophobic cluster. On the other hand, improper pairing of the cysteine residues in non-native single disulfide variants destabilizes the enzyme-inhibitor complex by inducing deformations of the binding loop region.

Correlation between disulfide reduction and conformational unfolding in bovine pancreatic trypsin inhibitor.

The native-like two-disulfide intermediate of bovine pancreatic trypsin inhibitor (BPTI), with the disulfide between Cys14 and Cys38 reduced, plays a particularly important role in the disulfide-coupled folding pathway of BPTI because of its participation in the rate-determining step of the reaction [Creighton & Goldenberg (1984) J. Mol. Biol. 179, 497-526; Weissman & Kim (1991) Science 253, 1386-1393]. In order to study directly the relationship between conformational stability and reductive unfolding kinetics, and to gain insight concerning the rate-limiting transition state in the thiol/disulfide-mediated folding/unfolding reaction of BPTI, BPTI variants based on a native-like two-disulfide analog of this intermediate, BPTI(Ala14)Ala38, were examined. The amino acid replacements introduced into BPTI(Ala14)Ala38 rendered it thermodynamically less stable. The kinetic stability, with respect to reduction by dithiothreitol, of the disulfides in these BPTI(Ala14)Ala38 variants was also decreased by the substitutions. The stabilization free energy (deltaG), obtained from chemical denaturation measurements, and the activation energy of the conformational transition (deltaG(++)conf), from the reductive unfolding reaction for this series of variants, were highly correlated. The observed correlation implies a direct coupling of disulfide reduction to conformational stability in this set of protein variants. It also strongly suggests that the transition state in the rate-limiting step of the reductive unfolding reaction involves a highly unfolded conformation of the protein. These data are consistent with a conformation-coupled redox folding pathway for BPTI(Ala14)Ala38 involving two parallel paths with unfolded (30-51) and unfolded (5-55) as the reactive species. Furthermore, the results provide a theoretical explanation for the observed 1000-fold diminution in the rate of 5-55 disulfide bond formation, relative to that of 14-38 bond formation, from the one-disulfide (30-51) intermediate in the wild-type BPTI refolding reaction. The data fit a general paradigm for protein disulfide formation during protein folding whereby native-like structure in folding intermediates accelerates formation of solvent-exposed disulfides but inhibits formation of core disulfides. This model predicts that a "rearrangement" mechanism (i.e., with non-native disulfides involved in the rate-limiting step) to form buried disulfides at a late stage in the folding reaction may be a common feature of redox folding pathways for surface disulfide-containing proteins of high stability.

Small effects of amino acid replacements on the reduced and unfolded state of pancreatic trypsin inhibitor.

The effects of amino acid replacements on the hydrodynamic volume of reduced and unfolded bovine pancreatic trypsin inhibitor (BPTI) have been examined by gel electrophoresis. The electrophoretic mobilities of the reduced forms of 46 BPTI variants were compared at room temperature in the absence of denaturants. The single substitutions examined include many different types of replacements at sites throughout the polypeptide, and, collectively, alter 22 of the 58 residues of the wild-type protein. The only substitutions found to alter the electrophoretic mobility of the reduced protein by more than approximately 3% are those that change the net charge of the protein. For nine mutants, the rates of disulfide formation in the reduced protein were also examined and found to be very similar to that of the wild-type protein. These results suggest that any structure that may be present in the reduced protein is either relatively insensitive to amino acid replacements or does not greatly influence the averaged properties of the polypeptide chain.

Intracellular expression of BPTI fusion proteins and single column cleavage/affinity purification by chymotrypsin

The novel and efficient expression system described here produces formerly poorly expressed, proteolytically unstable mutants of bovine pancreatic trypsin inhibitor (BPTI). A new, single column method for the cleavage of a recombinant fusion to BPTI and affinity purification of the BPTI moiety by immobilized chymotrypsin is an integral part of the system. Wild-type and mutant BPTI molecules are expressed in Escherichia coli as fusion proteins forming intracellular inclusion bodies. Transcription initiation is under the control of the E. coli trp promoter. The expressed protein is tripartite fusion comprising (i) a portion of the TrpLE leader peptide, (ii) a synthetic IgG binding domain derived from protein A and (iii) the BPTI variant. Solubilization of the inclusion bodies and refolding of the fusion proteins in a thiol-disulfide shuffling system yields correctly folded inhibitor molecules. In the single column purification and cleavage procedure, immobilized chymotrypsin cleaves the refolded fusion protein and releases affinity purified active BPTI mutants with correct N-termini. Mutant BPTI molecules which do not fold into active inhibitors are also stably expressed in inclusion bodies but cannot be purified by this method. Unlike previously described secretion systems for the production of BPTI, expression levels in this system appear to be independent of both the mutation in the BPTI gene and the activity of the expressed protein. Mutants poorly expressed in secretion systems can now be produced in sufficient quantities for protein folding studies and structural analysis using X-ray crystallography and NMR spectroscopy.

A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics

A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild-type protein. Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT). Under these conditions, unfolding and inactivation of the wild-type protein has a half-time of about 10 hours. Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate. Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated. The genes coding for 68 "DTT-sensitive" mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified. Most of the altered residues are largely buried in the core of the native wild-type structure and are highly conserved among proteins homologous to BPTI. These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated at most sites without completely preventing folding. Because this genetic screen is based on changes in folding energetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.