Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets

Indu, S. ; Kochat, V. ; Thakurela, S. ; Ramakrishnan, C. ; Varadarajan, Raghavan (2011) Conformational analysis and design of cross-strand disulfides in antiparallel β-sheets Proteins: Structure, Function,and Bioinformatics, 79 (1). pp. 244-260. ISSN 0887-3585

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Official URL: http://onlinelibrary.wiley.com/doi/10.1002/prot.22...

Related URL: http://dx.doi.org/10.1002/prot.22878

Abstract

Cross-strand disulfides bridge two cysteines in a registered pair of antiparallel β-strands. A nonredundant data set comprising 5025 polypeptides containing 2311 disulfides was used to study cross-strand disulfides. Seventy-six cross-strand disulfides were found of which 75 and 1 occurred at non-hydrogen-bonded (NHB) and hydrogen-bonded (HB) registered pairs, respectively. Conformational analysis and modeling studies demonstrated that disulfide formation at HB pairs necessarily requires an extremely rare and positive χ1 value for at least one of the cysteine residues. Disulfides at HB positions also have more unfavorable steric repulsion with the main chain. Thirteen pairs of disulfides were introduced in NHB and HB pairs in four model proteins: leucine binding protein (LBP), leucine, isoleucine, valine binding protein (LIVBP), maltose binding protein (MBP), and Top7. All mutants LIVBP T247C V331C showed disulfide formation either on purification, or on treatment with oxidants. Protein stability in both oxidized and reduced states of all mutants was measured. Relative to wild type, LBP and MBP mutants were destabilized with respect to chemical denaturation, although the sole exposed NHB LBP mutant showed an increase of 3.1°C in Tm. All Top7 mutants were characterized for stability through guanidinium thiocyanate chemical denaturation. Both exposed and two of the three buried NHB mutants were appreciably stabilized. All four HB Top7 mutants were destabilized (ΔΔG0 = −3.3 to −6.7 kcal/mol). The data demonstrate that introduction of cross-strand disulfides at exposed NHB pairs is a robust method of improving protein stability. All four exposed Top7 disulfide mutants showed mild redox activity.

Item Type:Article
Source:Copyright of this article belongs to John Wiley and Sons.
Keywords:Cross-strand Disulfides; NHB and HB Registered Pairs; MODIP; Torsion Angle; Chemical Denaturation; Thermostability
ID Code:57285
Deposited On:26 Aug 2011 04:21
Last Modified:11 Jul 2012 09:09

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