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

• Published in: Journal of molecular biology Vol. 369; no. 4; pp. 1087 - 1097
• Main Authors: Max, Klaas E.A., Wunderlich, Michael, Roske, Yvette, Schmid, Franz X., Heinemann, Udo
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 15-06-2007
• Subjects: Amino Acid Sequence; Bacillus subtilis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; cold shock protein; Cold Temperature; coulombic interactions; Crystallography, X-Ray; Directed Molecular Evolution; Genetic Variation; Models, Molecular; Molecular Sequence Data; Mutation; Proside in-vitro selection; Protein Denaturation; protein stability; Protein Structure, Tertiary; Sequence Alignment; Surface Properties; β-barrel; T M; Proside in-vitro selection; β-barrel; coulombic interactions; Bs-CspB; protein stability; cold shock protein
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2007.04.016

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 °C to 83.7 °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 β-barrel structure but not well optimized in the wild-type protein.