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

J Mol Biol. 2000 Apr 7;297(4):975-88. doi: 10.1006/jmbi.2000.3602.


The bacterial cold shock proteins are small compact beta-barrel proteins without disulfide bonds, cis-proline residues or tightly bound cofactors. Bc-Csp, the cold shock protein from the thermophile Bacillus caldolyticus shows a twofold increase in the free energy of stabilization relative to its homolog Bs-CspB from the mesophile Bacillus subtilis, although the two proteins 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 cooperative transitions with T(M) values of 49.0 degrees C and 77.3 degrees C, respectively, at pH 7.0. Addition of 0.5 M salt stabilizes Bs-CspB but destabilizes Bc-Csp. To understand these differences at the structural level, the crystal structure of Bc-Csp was determined at 1.17 A resolution and refined to R=12.5% (R(free)=17.9%). The molecular structures of Bc-Csp and Bs-CspB are virtually identical in the central beta-sheet and in the binding region for nucleic acids. Significant differences are found in the distribution 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 unfavorable for Bs-CspB. They provide the major source for the increased thermostability of Bc-Csp. This can be explained based on the atomic-resolution crystal structure of Bc-Csp. It identifies a number of potentially stabilizing ionic interactions including a cation-binding site and reveals significant changes in the electrostatic surface potential.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillus / chemistry*
  • Bacterial Proteins / chemistry*
  • Binding Sites
  • Cations / metabolism
  • Cations / pharmacology
  • Circular Dichroism
  • Crystallography, X-Ray
  • Heat-Shock Proteins / chemistry*
  • Heat-Shock Proteins / metabolism*
  • Hot Temperature
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acids / metabolism
  • Osmolar Concentration
  • Protein Denaturation / drug effects
  • Protein Folding
  • Protein Structure, Secondary / drug effects
  • Protein Structure, Tertiary / drug effects
  • Sodium / metabolism
  • Sodium / pharmacology
  • Static Electricity
  • Structure-Activity Relationship
  • Thermodynamics


  • Bacterial Proteins
  • Cations
  • Heat-Shock Proteins
  • Nucleic Acids
  • cold shock protein, Bacillus
  • cold-shock protein CspB, Bacteria
  • Sodium

Associated data

  • PDB/1C90