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D P Goldenberg

Publications and source records attributed to D P Goldenberg.

At least 37 records · Page 2Linked to original sources
PubMed1993

Probing the determinants of disulfide stability in native pancreatic trypsin inhibitor.

The effects of single amino acid replacements on the stability of the 14-38 disulfide bond in the native form of bovine pancreatic trypsin inhibitor (BPTI) were measured. A total of 17 mutant proteins, with substitutions at one of 7 residues located 5-15 A from the disulfide in the native wild-type protein, were examined. The replacements were found to decrease the thermodynamic stability of the disulfide, as measured by exchange with thiol-disulfide reagents, by 0.6-5 kcal/mol, corresponding to a range of nearly 100 mV in redox potentials. The effects of the substitutions on disulfide stability were roughly correlated with the changes in side-chain volume, suggesting that optimal packing is a major factor in determining the stability of the disulfide in the wild-type protein. With only one exception, the substitutions also led to increases, as large as 50-fold, in the rates of disulfide reduction by dithiothreitol. The increased rates of reduction suggest that at least a fraction of the mutational destabilization of the disulfide is due to strain in the native protein that is relieved in the transition state for reduction. The stability of the disulfide in a peptide corresponding to the segments that are linked together by the 14-38 disulfide in native BPTI was found to be about 5 kcal/mol less than that of the disulfide in the intact wild-type protein. Together, the results with the mutant proteins and the peptide indicate that the stability of the disulfide in the native protein depends on both the local environment of the disulfide and on the ability of the rest of the protein to favor a conformation that promotes disulfide formation.

Amino Acid Sequence↗
PubMed1993

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.

Amino Acids↗
PubMed1992

Sequential translocation of an artificial precursor protein across the two mitochondrial membranes.

We have constructed a chimeric mitochondrial precursor protein consisting of a mutant bovine pancreatic trypsin inhibitor coupled to the C terminus of a purified artificial precursor protein. This construct fails to complete its import into isolated mitochondria and becomes stuck across sites of close contact between the two mitochondrial membranes. When the mitochondria are then depleted of ATP and the intramolecular disulfide bridges of the trypsin inhibitor are cleaved by dithiothreitol, the trypsin inhibitor moiety is transported across the outer membrane into the intermembrane space. This translocation intermediate can be chased across the inner membrane by restoring the ATP levels in the matrix. These results show that translocation of pancreatic trypsin inhibitor across a biological membrane is prevented by its intramolecular disulfide bridges, that import into the matrix involves two distinct translocation system operating in tandem, and that ATP is required for protein translocation across the inner but not the outer membrane.

Adenosine Triphosphate↗
PubMed1992

Genetic dissection of pancreatic trypsin inhibitor.

In a previous study, a genetic screening procedure was used to identify variants of bovine pancreatic trypsin inhibitor that can fold to an active conformation but that are inactivated much more rapidly than the wild-type protein in the presence of dithiothreitol (DTT). The mechanisms by which 30 of these DTT-sensitive variants are inactivated have now been investigated. Some of the amino acid replacements cause rapid inactivation in the presence of DTT because the three disulfides of the native protein are reduced up to 300-fold faster than for the wild-type protein, leading to complete unfolding. Other substitutions, however, do not greatly increase the rate of complete reduction and unfolding but lead to accumulation of an inactive two-disulfide species. There is a striking correlation between the locations of the DTT-sensitive amino acid replacements in the three-dimensional structure of the protein and the mechanisms by which the variants are inactivated. All of the substitutions that cause rapid unfolding are clustered at one end of the folded protein, in the vicinity of the two disulfides that are reduced most slowly during unfolding of the wild-type protein, while substitutions of the other class are all located at the other end of the protein, near the trypsin binding site. These results indicate that the kinetic stability of native bovine pancreatic trypsin inhibitor and its ability to function as a protease inhibitor are largely influenced by residues in two distinguishable regions of the folded protein.

Amino Acid Sequence↗
PubMed1992

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.

Animals↗
PubMed1991

Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates.

The thermodynamics and kinetics of unfolding of 28 bacteriophage T4 lysozyme variants were compared by using urea gradient gel electrophoresis. The mutations studied cause a variety of sequence changes at different residues throughout the polypeptide chain and result in a wide range of thermodynamic stabilities. A striking relationship was observed between the thermodynamic and kinetic effects of the amino acid replacements: All the substitutions that destabilized the native protein by 2 kcal/mol or more also increased the rate of unfolding. The observed increases in unfolding rate corresponded to a decrease in the activation energy of unfolding (delta Gu) at least 35% as large as the decrease in thermodynamic stability (delta Gu). Thus, the destabilizing lesions bring the free energy of the native state closer to that of both the unfolded state and the transition state for folding and unfolding. Since a large fraction of the mutational destabilization is expressed between the transition state and the native conformation, the changes in folding energetics cannot be accounted for by effects on the unfolded state alone. The results also suggest that interactions throughout much of the folded structure are altered in the formation of the transition state during unfolding.

Crystallography↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1989

Mutational analysis of a protein-folding pathway.

The effects of amino-acid replacements on the disulphide-coupled folding pathway of bovine pancreatic trypsin inhibitor have been examined. Replacements at three sites destabilize the native protein relative to the unfolded state, but have different effects on the relative stabilities of the disulphide-bonded folding intermediates, thus allowing the roles of the altered residues during folding to be distinguished.

Amino Acid Sequence↗
PubMed1988

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].

Amino Acid Sequence↗
PubMed1988

Genetic studies of protein stability and mechanisms of folding.

In the past few years, analysis of mutationally altered proteins has joined more traditional biophysical methods as an important experimental approach to the study of protein folding. Single amino acid substitutions have been found to reduce the stability of the native state by as much as 3 kcal mol-1, a significant fraction of the marginal stability of the folded conformation, and to significantly alter the rates of folding or unfolding. Mutations can also significantly affect the ability of a protein to fold in vivo. However, at present there is great uncertainty in accounting for or predicting the effects of mutations on stability and folding, even qualitatively. Future studies of mutant proteins are likely to provide further insights into the roles of individual residues and interactions in determining the three-dimensional structures of proteins. For the full potential of this approach to be realized, however, many mutant proteins will have to be systematically studied using structural, thermodynamic, and kinetic methods. The results of these experiments may aid in developing and refining improved theoretical treatments of the energetics of protein conformation.

Drug Stability↗
PubMed1985

Comparative studies of conformation and internal mobility in native and circular basic pancreatic trypsin inhibitor by 1H nuclear magnetic resonance in solution.

A circular derivative of bovine pancreatic trypsin inhibitor (c-BPTI) with the C- and N-termini covalently linked with a peptide bond, has been studied in solution by spectroscopic techniques, principally two-dimensional 1H NMR. Near complete sequence specific assignment of the 1H NMR spectrum of c-BPTI has been obtained in a highly efficient manner, using recently developed experimental techniques. The assignments serve as the necessary background for detailed examination of the effects of the chemical modification on global and local features of the protein conformation and on the internal mobility. Analysis of chemical shifts, spin-spin coupling constants, amide proton exchange and proton-proton Overhauser enhancements indicates that perturbations of the native BPTI conformation in c-BPTI involve exclusively residues adjacent to the modification site. The strict localization and small magnitude of the conformation changes relative to native BPTI emphasize the important role of the salt bridge between the chain ends in determining the solution conformation and dynamics in the chain terminal regions of the native protein.

Amides↗
PubMed1985

Dissecting the roles of individual interactions in protein stability: lessons from a circularized protein.

A circular form of bovine pancreatic trypsin inhibitor (BPTI) has been prepared by introducing a peptide bond between the N- and C-termini, which are in close proximity in the native conformation. The pathway and energetics of the disulphide-coupled folding transition of the circular protein have been studied using methods applied previously to the unmodified protein. The cross-link between the termini was found not to significantly stabilize the native state in spite of the expected reduction in entropy of the unfolded protein. This unexpected result has led to a reexamination of the stabilization expected from a cross-link, considering effects on the native, as well as unfolded, states of the protein. The greatest stabilization is expected when the cross-linked groups are held rigidly in the native protein in the optimum orientation for forming the cross-link. Similar analyses, utilizing thermodynamic cycles, can be applied to other interactions that stabilize native proteins, including disulphide bonds, salt bridges, and hydrogen bonds and to modifications to the protein that remove them. In general, the contribution of an individual interaction to the stability of the native state depends on the extent to which the interaction is favored in the native conformation, which can vary greatly depending on the local environment of the interacting groups.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

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.

Animals↗
PubMed1984

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.

Animals↗
PubMed1983

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.

Amino Acid Sequence↗