Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor

Pan, Hong; Barbar, Elisar; Bárány, George; Woodward, Clare

doi:10.1021/bi00043a002

Cited by 67 publications

(58 citation statements)

References 25 publications

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“…The farultraviolet CD spectrum of the reduced protein is very similar to those seen for other unfolded proteins and indicates an absence of regular secondary structure~Kosen et al, 1981!. On the other hand, NMR studies of reduced BPTI and peptide fragments thereof have demonstrated the presence of numerous specific, but probably transient, interactions, and local conformational preferences Lumb & Kim, 1994;Pan et al, 1995!. Interestingly, many of the interactions detected by NMR are not present in the native protein, and it has been suggested that forming any of the three native disulfides causes the disruption of these non-native interactions~Pan et al, 1995!.…”

Section: The Nature and Role Of Nonrandom Structure In Fully Reduced mentioning

confidence: 55%

Early events in the disulfide‐coupled folding of BPTI

Bułaj

Goldenberg

1999

Protein Science

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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 disulfideformation 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 8C 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.Keywords: @14-38# intermediate; bovine pancreatic trypsin inhibitor; disulfide bonds; protein folding; unfolded proteins For more than two decades, bovine pancreatic trypsin inhibitor BPTI! has served as an important model for both theoretical and experimental studies of protein folding~Levitt & Warshel, 1975;Creighton, 1978;Goldenberg et al., 1989;Weissman & Kim, 1991;Goldenberg, 1992;Daggett & Levitt, 1993;Darby et al., 1995!. Pioneering work by Creighton~1978! demonstrated that the folded structure of BPTI depends on its three disulfide bonds and that these disulfides could be used as experimental probes of conformation during oxidative refolding of the reduced protein. These experiments revealed that only a fraction of the possible disulfidebonded intermediates accumulate to significant levels during refolding, thus providing some of the first experimental evidence to support the view that proteins fold via specific pathways. In subsequent studies, a variety of techniques, including NMR spectroscopy and mutational analysis, have been used to analyze the conformations of the intermediates and the interactions that stab...

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Section: The Nature and Role Of Nonrandom Structure In Fully Reduced mentioning

confidence: 55%

Early events in the disulfide‐coupled folding of BPTI

Bułaj

Goldenberg

1999

Protein Science

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“…Our results show that the denatured ensemble of BPTI is not a fully random coil but collapsed with some ordered structure. 69,70 There are several interesting differences among the denatured states. Unfolded reduced BPTI with all three disulfide bonds broken sample non-native conformations that are absent in the unfolded 14 -38 BPTI analogues.…”

Section: Denatured Ensemblementioning

confidence: 99%

NMR characterization of partially folded and unfolded conformational ensembles of proteins

Barbar

1999

Biopolymers

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Studies of unfolded and partially folded proteins provide important insight into the initiation and process of protein folding. This review focuses on the use of nmr in characterization of ensembles of proteins that model the early stages of folding. Analysis of an ensemble includes description of the number of conformations, their structure, relative populations, interconversion rates, and dynamics of subconformations. A chemically synthesized analogue of bovine pancreatic trypsin inhibitor (BPTI), [14–38]Abu, has provided a rare system for characterization of multiple partially folded conformations in slow exchange at near physiological conditions. Multidimensional nmr techniques coupled with selective labeling were used to probe different segments of the polypeptide chain. At each labeled site, there is evidence of slow interconversion between two families of partially folded conformations that in themselves are ensembles of rapidly interconverting conformers. All these conformers display significantly more order in the core relative to the rest of the molecule. For other variants of BPTI that are unfolded at equilibrium, the most ordered structure is also favored in the hydrophobic core residues of the native protein. This is consistent with the hypothesis that the residues that are the first to fold go on to form the most stable, structure‐determining part of the protein. © 1999 John Wiley & Sons, Inc. Biopoly 51: 191–207, 1999

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“…It is only recently that structural information for the denatured state has become available at atomic resolution because of developments in high field NMR spectroscopy (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). These studies have focused on areas of persistent structure, with the aim of obtaining insight into the relationship between sequence, structure, and in particular, the mechanism of folding.…”

Section: Introductionmentioning

confidence: 99%

Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: Description of the folding pathway

Bond

Wong

Clarke

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

123

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one of the authors regrets that she inadvertently omitted references to the computer program and protein potential function that the authors used for their simulations of barnase cited above. The following sentence should have been the first sentence of the Methods section: Molecular dynamics simulations were performed with the program ENCAD (44) and the potential energy function of Levitt et al. (45).

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Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor

Cited by 67 publications

References 25 publications

Early events in the disulfide‐coupled folding of BPTI

Early events in the disulfide‐coupled folding of BPTI

NMR characterization of partially folded and unfolded conformational ensembles of proteins

Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: Description of the folding pathway

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