Protein folding guides disulfide bond formation

Abstract

The Anfinsen principle that the protein sequence uniquely determines its structure is based on experiments on oxidative refolding of a protein with disulfide bonds. The problem of how protein folding drives disulfide bond formation is poorly understood. Here, we have solved this long-standing problem by creating a general method for implementing the chemistry of disulfide bond formation and rupture in coarse-grained molecular simulations. As a case study, we investigate the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI). After confirming the experimental findings that the multiple routes to the folded state contain a network of states dominated by native disulfides, we show that the entropically unfavorable native single disulfide between Cys 14 and Cys 38 forms only after polypeptide chain collapse and complete structuring of the central core of the protein containing an antiparallel β-sheet. Subsequent assembly, resulting in native two-disulfide bonds and the folded state, involves substantial unfolding of the protein and transient population of nonnative structures. The rate of formation increases as the β-sheet stability increases. The flux to the native state, through a network of kinetically connected native-like intermediates, changes dramatically by altering the redox conditions. Disulfide bond formation between Cys residues not present in the native state are relevant only on the time scale of collapse of BPTI. The finding that formation of specific collapsed native-like structures guides efficient folding is applicable to a broad class of singledomain proteins, including enzyme-catalyzed disulfide proteins.disulfide proteins | native-like interactions | enzyme-catalyzed folding | early collapse | nonnative interactions T he landmark discovery that the information to fold a protein is fully contained in the primary amino acid sequence was based on oxidative refolding experiments on disulfide bond formation in ribonuclease A (RNase A) (1, 2). Anfinsen showed that the initially unfolded protein, generated by reducing the disulfide (S-S) bonds in the native state of RNase A, folds reversibly under oxidizing conditions by correctly reforming the four native S-S bonds (among 105 possibilities) between the eight cysteine (Cys) residues. Besides being central to the enunciation of the principles of protein folding, many secretary proteins, whose misfolding is linked to a number of diseases, contain S-S bonds (3). Although biophysical aspects of such proteins are not as well studied as those without S-S bonds, understanding the link between conformational folding coupled to disulfide bond formation (4-7) is important and challenging both from a chemical and biophysical perspective (8).The formation of S-S bonds and their identities during folding can be monitored by quenching the oxidative process at various stages of the folding reaction (9). By arresting the reaction, it is possible to characterize the accumulated intermediates in terms of already formed S-S bonds (10). However, the relatio...

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