David P. Goldenberg

Kinetic role of a meta-stable native-like two-disulphide species in the folding transition of bovine pancreatic trypsin inhibitor

The properties have been determined of a recently identified form of bovine pancreatic trypsin inhibitor, with only two of the three disulphide bonds of the native protein, but possessing a native-like conformation. The kinetics of refolding of the reduced inhibitor were re-measured to elucidate the kinetic role in folding of this two-disulphide species; it is formed both directly from a minor one-disulphide intermediate and by rearrangement of the other two-disulphide intermediates. It is not a productive intermediate because the Cys30 and Cys51 thiols are buried and unreactive. The previous kinetic analysis was extended by using both intra- and intermolecular disulphide reagents. Entirely consistent kinetic parameters for the rates of all the intramolecular steps of the pathway were obtained, and use of both types of reagents permits a detailed dissection of the kinetic pathway. In the process, the energetics of the folding transition were measured more thoroughly. The unique information available about the formation and stabilities of the disulphides during refolding of reduced bovine pancreatic trypsin inhibitor provides a useful description of the way in which numerous weak interactions within a protein co-operate to produce a stable folded conformation.

Circular and circularly permuted forms of bovine pancreatic trypsin inhibitor

Two novel forms of bovine pancreatic trypsin inhibitor have been prepared. The amino- and carboxyl-termini, which are in close proximity in the native conformation, were linked together in a peptide bond, thus generating a molecule with a circular backbone. The circular molecule was then cleaved between Lys15 and Ala16, to yield a linear molecule whose sequence is a circular permutation of that of bovine pancreatic trypsin inhibitor. Both of these modified forms could refold to the native conformation after being reduced, and promise to be interesting subjects for further folding experiments.

Gel electrophoresis in studies of protein conformation and folding.

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.

Computational Simulation of the Statistical Properties of Unfolded Proteins

A simple Monte Carlo method was used to generate ensembles of simulated polypeptide conformations that are restricted only by steric repulsion. The models used for these simulations were based on the sequences of four real proteins, ranging in size from 26 to 268 amino acid residues, and included all non-hydrogen atoms. Two sets of calculations were performed, one that included only intra-residue steric repulsion terms and those between adjacent residues, and one that included repulsion terms between all possible atom pairs, so as to explicitly account for the excluded volume effect. Excluded volume was found to increase the average radius of gyration of the chains by 20-40%, with the expansion factor increasing with chain length. Contrary to recent suggestions, however, the excluded volume effect did not greatly restrict the distribution of dihedral angles or favor native-like topologies. The average dimensions of the ensembles calculated with excluded volume were consistent with those measured experimentally for unfolded proteins of similar sizes under denaturing conditions, without introducing any adjustable scaling factor. The simulations also reproduced experimentally determined effective concentrations for the formation of disulfide bonds in reduced and unfolded proteins. The statistically generated ensembles included significant numbers of conformations that were nearly as compact as the corresponding native proteins, as well as many that were as accessible to solvent as a fully extended chain. On the other hand, conformations with as much buried surface area as the native proteins were very rare, as were highly extended conformations. These results suggest that the overall properties of unfolded proteins can be usefully described by a random coil model and that an unfolded polypeptide can undergo significant collapse while losing only a relatively small fraction of its conformational entropy.

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.

Temperature-sensitive mutants blocked in the folding or subunit assembly of the bacteriophage P22 tail spike protein: II. Active mutant proteins matured at 30 °C☆☆☆

Abstract Little is known of the molecular mechanisms by which temperature-sensitive mutations interfere with the formation of biologically active proteins. We have studied the effects of such mutations at 13 different sites on the properties of the multifunctional tail spike protein of bacteriophage P22, a thermostable structural protein composed of 76,000 M r chains. Using multiple mutant strains blocked in capsid assembly, we have examined the free mutant tail spikes that accumulate in active form at permissive temperature. When assayed for the ability to bind to phage heads at the restrictive temperature, the mutant proteins were as active as the wild type. Similarly, when assayed for the ability to adsorb to bacteria at restrictive temperature, the mutant proteins were as active as the wild type. Thus the temperature-sensitive phenotypes of the mutants are not due to the thermolability of these functions in the mature mutant protein. The wild-type protein is heat-resistant, requiring incubation at 90 °C, to give a half-time of inactivation of ten minutes. The 13 ts mutant proteins, once matured at 30 °C, were as resistant as the wild-type protein to inactivation at elevated temperatures. Though the mature wild-type protein is heat stable, its maturation is heat-sensitive; the number of polypeptide chains synthesized at 30 °C and 39 °C is the same, but the yield of active tail spikes at 39 °C is only 25% of the yield at 30 °C. The results show that the amino acid substitutions in the mutant proteins, though lethal for the formation of the virus at 39 °C, do not affect the thermostability of the mature tail spike protein formed at 30 °C. They may act by destabilizing thermolabile intermediates in the folding or subunit assembly of the tail spike protein.

Finding the right fold

Using mutational analysis, three groups have compared the transition states for the folding of two pairs of homologous proteins. The results of these studies suggest that protein folding mechanisms are conserved and are defined primarily by the overall topology of the native structures, as opposed to specific details of the interactions stabilizing these structures.

Folding pathway of a circular form of bovine pancreatic trypsin inhibitor

The pathway of unfolding and refolding of a circular form of bovine pancreatic trypsin inhibitor, in which the termini were linked together in a peptide bond, has been examined by trapping and identifying the disulphide-containing intermediates, as was done previously for the unmodified protein. The folding pathway of the circular protein was essentially the same as that of the unmodified inhibitor, although there were differences in the distribution of intermediates that accumulated and in the rates of some steps. The effects of the cross-link between the termini on the stabilities of the folding intermediates and the native state were determined by measuring the rates of the interconversions making up the folding transition, and comparing them with those measured for the unmodified protein. The major effect of the cross-link was to stabilize an intermediate containing two native disulphides, (30-51, 14-38), but lacking the disulphide nearest the termini, 5-55. The native conformation was not measurably stabilized by the cross-link, in spite of the expected reduction of entropy of the unfolded state, indicating that the native state of the circular protein had a slightly strained conformation. The stabilities of the major one-disulphide intermediates were not significantly affected by the cross-link, suggesting that the termini of bovine pancreatic trypsin inhibitor do not tend to interact during the early stage of folding.

Effects of Macromolecular Crowding on an Intrinsically Disordered Protein Characterized by Small-Angle Neutron Scattering with Contrast Matching

Small-angle neutron scattering was used to examine the effects of molecular crowding on an intrinsically disordered protein, the N protein of bacteriophage λ, in the presence of high concentrations of a small globular protein, bovine pancreatic trypsin inhibitor (BPTI). The N protein was labeled with deuterium, and the D(2)O concentration of the solvent was adjusted to eliminate the scattering contrast between the solvent and unlabeled BPTI, leaving only the scattering signal from the unfolded protein. The scattering profile observed in the absence of BPTI closely matched that predicted for an ensemble of random conformations. With BPTI added to a concentration of 65 mg/mL, there was a clear change in the scattering profile representing an increase in the mass fractal dimension of the unfolded protein, from 1.7 to 1.9, as expected if crowding favors more compact conformations. The crowding protein also inhibited aggregation of the unfolded protein. At 130 mg/mL BPTI, however, the fractal dimension was not significantly different from that measured at the lower concentration, contrary to the predictions of models that treat the unfolded conformations as convex particles. These results are reminiscent of the behavior of polymers in concentrated melts, suggesting that these synthetic mixtures may provide useful insights into the properties of unfolded proteins under crowding conditions.

Structure of a serine protease poised to resynthesize a peptide bond

The serine proteases are among the most thoroughly studied enzymes, and numerous crystal structures representing the enzyme–substrate complex and intermediates in the hydrolysis reactions have been reported. Some aspects of the catalytic mechanism remain controversial, however, especially the role of conformational changes in the reaction. We describe here a high-resolution (1.46 Å) crystal structure of a complex formed between a cleaved form of bovine pancreatic trypsin inhibitor (BPTI) and a catalytically inactive trypsin variant with the BPTI cleavage site ideally positioned in the active site for resynthesis of the peptide bond. This structure defines the positions of the newly generated amino and carboxyl groups following the 2 steps in the hydrolytic reaction. Comparison of this structure with those representing other intermediates in the reaction demonstrates that the residues of the catalytic triad are positioned to promote each step of both the forward and reverse reaction with remarkably little motion and with conservation of hydrogen-bonding interactions. The results also provide insights into the mechanism by which inhibitors like BPTI normally resist hydrolysis when bound to their target proteases.