title: Origins of the high stability of an in vitro-selected cold-shock protein.
abstract: In previous work, we had identified stabilized forms of the cold-shock protein Bs-CspB from Bacillus subtilis in a combinatorial library by an in vitro selection procedure. In this library, the sequence positions 2, 3, 46, 64, 66, and 67 had been randomized, because Bs-CspB differs from the naturally thermostable homolog Bc-Csp from Bacillus caldolyticus, among others, at these six positions. For the most stable selected variant, the midpoint of thermal unfolding (tM) increased by 28.2 deg. C and the Gibbs free energy of unfolding (deltaG(D)) by 19 kJ/mol. Here, we analyzed by site-directed mutagenesis how the selected residues contribute individually to this strong stabilization. Val3 and Val66, which replace Glu3 and Glu66 of wild-type Bs-CspB, each contribute about 7 kJ/mol to stability, the Thr64Arg substitution contributes 4.5 kJ/mol, and 3.2 kJ/mol originate from the Ala46Leu replacement. Gly67 at the carboxy terminus is unimportant for stability, the Arg selected at position 2 is overall slightly destabilizing but improves the coulombic interactions. The best variant differs from Bc-Csp at all six positions; nevertheless, natural and in vitro selection followed similar principles. In both cases, negatively charged residues at the adjacent positions 3 and 66 are avoided, and a positively charged residue is introduced into this area of the protein surface. Its exact location is unimportant. It can be at position 3, as in the thermophilic Bc-Csp, or at positions 2 or 64, as in the most stable selected variant. These positively charged residues contribute to stability not by engaging in pairwise coulombic interactions with a specific carboxyl group, but by generally improving the charge distribution in this particular region of the protein surface. These coulombic effects contribute significantly to the thermostability of the cold-shock proteins. They are only weakly interdependent and best explained by the presence of a flexible ion network at the protein surface. Our results emphasize that surface positions are very good candidates for optimizing protein stability.
authors.affiliation: Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Martin
authors.forename: Insa
authors.initials: I
authors.lastname: Kather
authors.forename: Franz X
authors.initials: FX
authors.lastname: Schmid
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D019151:Peptide Library; C407094:cold shock protein, Bacillus