Structural determinants of oxidative folding in proteins

Citation
E. Welker et al., Structural determinants of oxidative folding in proteins, P NAS US, 98(5), 2001, pp. 2312-2316
Citations number
31
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN journal
00278424 → ACNP
Volume
98
Issue
5
Year of publication
2001
Pages
2312 - 2316
Database
ISI
SICI code
0027-8424(20010227)98:5<2312:SDOOFI>2.0.ZU;2-X
Abstract
A method for determining the kinetic fate of structured disulfide species ( i.e., whether they are preferentially oxidized or reshuffle back to an unst ructured disulfide species) is introduced. The method relies on the sensiti vity of unstructured disulfide species to low concentrations of reducing ag ents. Because a structured des species that preferentially reshuffles gener ally first rearranges to an unstructured species, a small concentration of reduced DTT (e.g., 260 muM) suffices to distinguish on-pathway intermediate s from dead-end species. We apply this method to the oxidative folding of b ovine pancreatic ribonuclease A (RNase A) and show that des[40-95] and des[ 65-72] are productive intermediates, whereas des[26-84] and des[58-110] are metastable dead-end species that preferentially reshuffle. The key factor in determining the kinetic fate of these des species is the relative access ibility of both their thiol groups and disulfide bonds. Productive intermed iates tend to be disulfide-secure, meaning that their structural fluctuatio ns preferentially expose their thiol groups, while keeping their disulfide bonds buried. By contrast, dead-end species tend to be disulfide-insecure, in that their structural fluctuations expose their disulfide bonds in conce rt with their thiol groups. This distinction leads to four generic types of oxidative folding pathways. We combine these results with those of earlier studies to suggest a general three-stage model of oxidative folding of RNa se A and other single-domain proteins with multiple disulfide bonds.