Optimized variants of the cold shock protein from in vitro selection: structural basis of their high thermostability.
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.
Max
Klaas E A
KE
Makromolekulare Strukturen und Interaktionen, Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.
Wunderlich
Michael
M
Roske
Yvette
Y
Schmid
Franz X
FX
Heinemann
Udo
U
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003080:Cold Temperature; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D014644:Genetic Variation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013499:Surface Properties
D001426:Bacterial Proteins; C077188:cold-shock protein CspB, Bacteria