Thermal stability and atomic-resolution crystal structure of the Bacillus caldolyticus cold shock protein

The bacterial cold shock proteins are small compact beta-barrel proteins wi thout disulfide bonds, cis-proline residues or tightly bound cofactors. Bc- Csp, the cold shock protein from the thermophile Bacillus caldolyticus show s a twofold increase in the free energy of stabilization relative to its ho molog Bs-CspB from the mesophile Bacillus subtilis, although the two protei ns differ by only 12 out of 67 amino acid residues. This pair of cold shock proteins thus represents a good system to study the atomic determinants of protein thermostability. Bs-CspB and Bc-Csp both unfold reversibly in coop erative transitions with T-M values of 49.0 degrees C and 77.3 degrees C, r espectively, at pH 7.0. Addition of 0.5 M salt stabilizes Bs-CspB but desta bilizes Bc-Csp. To understand these differences at the structural level, th e crystal structure of Bc-Csp was determined at 1.17 Angstrom resolution an d refined to R = 12.5 % (R(free) = 17.9 %). The molecular structures of Bc- Csp and Bs-CspB are virtually identical in the central P-sheet and in the b inding region for nucleic acids. Significant differences are found in the d istribution of surface charges including a sodium ion binding site present in Bc-Csp, which was not observed in the crystal structure of the Bs-CspB. Electrostatic interactions are overall favorable for Bc-Csp, but unfavorabl e for Bs-CspB. They provide the major source for the increased thermostabil ity of Bc-Csp. This can be explained based on the atomic-resolution crystal structure of Bc-Csp. It identifies a number of potentially stabilizing ion ic interactions including a cation-binding site and reveals significant cha nges in the electrostatic surface potential. (C) 2000 Academic Press.