A look at: “Disulfide Bonds and Protein Folding”

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In this series, we are highlighting some of our founder, Dr. Yelena Sheptovitsky’s, favorite manuscripts, publications, and news pieces all relating to the world of protein expression, life science, and CRO’s.

Today we look at the author manuscript from “Disulfide Bonds and Protein Folding” by William J. Wedemeyer, Ervin Welker, Mahesh Narayan, and Harold A. Scheraga* Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301 Received December 21, 1999; Revised Manuscript Received February 9, 2000

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The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of “rugged funnel” models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulfide bonds suggests an analogous scenario for conformational folding. Finally, engineered covalent cross-links may be used to assay for the association of protein segments in the folding transition state, as illustrated with RNase A.

One area that stands to be mentioned:


Disulfide bonds are useful tools for studying protein structure, thermodynamics, and folding. In particular, disulfide bonds may be used to assess the accessibility, proximity, and reactivity of cysteine residues. For example, the reductive unfolding of the protein reports on structural fluctuations in the native state because the protein must undergo local or global unfolding to render buried disulfide bonds accessible to the redox reagent. The proximity assay of disulfide bonds has been used to study â-hairpin propensities (102-104), the tertiary fold of the molten globule (55), and the rotation of molecular motors composed of many proteins (105). More generally, the distribution of disulfide-bonded species and the rates of their individual formation provide information about the conformational order in the protein; a disordered protein should show a random distribution of disulfide species (determined primarily by the entropy loss upon forming the disulfide bonds), whereas a highly ordered or structured protein should exhibit a strongly nonrandom distribution of disulfide species and bonds. As another example, the contribution of individual disulfide bonds to the conformational stability of the native state can often be assessed by reducing a single disulfide bond; provided that the resulting des species remains folded, its thermodynamic stability can be examined by equilibrium unfolding transitions and by the H/D exchange properties of its residues (25, 26, 74).

The oxidative folding of RNase A can be divided into two stages. In the first stage, the reduced protein is successively oxidized to populate the 1S-4S ensembles. These ensembles have no stable tertiary structure, although they exhibit conformational ordering, e.g., a nonrandom distribution of disulfide species. The second stage of oxidative folding consists of the disulfide reactions in the structured disulfide-containing intermediates that are stable enough to maintain their folded conformation (des species). The resulting tertiary structure stabilizes these folded species by protecting their native disulfide bonds from reduction and reshuffling; these structured species do not interconvert readily among themselves or with their unstructured ensemble. Under typical oxidative folding conditions, only des[40-95] and des[65-72] are stable enough to fold; these species are productive intermediates, since their remaining thiol groups are accessible, allowing these species to oxidize preferentially to the native protein. However, under unusually stabilizing conditions, des[26-84] and des[58-110] species can also acquire stable tertiary structure. When folded, these latter species, accounting for only a minor percentage of the total protein, appear to be kinetic traps, and presumably bury both their disulfide bonds and thiol groups, rendering all the reactive groups inaccessible to the redox reagent and to each other. Such traps may preferentially oxidize, reshuffle, or be reduced, depending on the redox conditions and the relative free energies of the unfolding reactions that must precede each reaction.

The processes of reductive unfolding and oxidative folding and the conformational structure of disulfide intermediates have several implications for conformational folding. The evidence seems to support multinucleation models of protein folding such as the CFIS model and to contradict the recently proposed “rugged funnel” scenarios of protein folding. By analogy with the regeneration of the native disulfide bonds, it is suggested that conformational folding proceeds through a biased random search of an initially unstructured ensemble, leading to the formation of a densely packed core of native structure that is protected from attack by the solvent and competing groups of the protein; this core then promotes the formation of the remaining native structure. The central idea of this scenario is that certain native structures become favored, not because they form much more quickly than non-native structures, but chiefly because they rearrange much more slowly.


To read the full manuscript, click here.


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