Optimized Variants of the Cold Shock Protein from in Vitro Selection: Structural Basis of Their High Thermostability

Item Type:	Article
Title:	Optimized Variants of the Cold Shock Protein from in Vitro Selection: Structural Basis of Their High Thermostability
Creators Name:	Max, K.E. and Wunderlich, M. and Roske, Y. and Schmid, F.X. and Heinemann, U.
Abstract:	The bacterial cold shock proteins (Csp) are widely used as models for the experimental and computational analysis of protein stability. In a previous study, in vitro evolution was employed to identify strongly stabilizing mutations in Bs-CspB from Bacillus subtilis. The best variant found by this approach contained the mutations M1R, E3K and K65I, which raised the midpoint of thermal unfolding of Bs-CspB from 53.8 degrees C to 83.7 degrees C, and increased the Gibbs free energy of stabilization by 20.9 kJ mol(-1). Another selected variant with the two mutations A46K and S48R was stabilized by 11.1 kJ mol(-1). To elucidate the molecular basis of these stabilizations, we determined the crystal structures of these two Bs-CspB variants. The mutated residues are generally well ordered and provide additional stabilizing interactions, such as charge interactions, additional hydrogen bonds and improved side-chain packing. Several mutations improve the electrostatic interactions, either by the removal of unfavorable charges (E3K) or by compensating their destabilizing interactions (A46K, S48R). The stabilizing mutations are clustered at a contiguous surface area of Bs-CspB, which apparently is critically important for the stability of the beta-barrel structure but not well optimized in the wild-type protein.
Keywords:	Beta-Barrel, Cold Shock Protein, Soulombic Interactions, Proside Invitro Selection, Protein Stability
Source:	Journal of Molecular Biology
ISSN:	0022-2836
Publisher:	Elsevier
Volume:	369
Number:	4
Page Range:	1087-1097
Date:	15 June 2007
Official Publication:	https://doi.org/10.1016/j.jmb.2007.04.016
PubMed:	View item in PubMed

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