title: Mechanism of thermostabilization in a designed cold shock protein with optimized surface electrostatic interactions.
abstract: Using computational and sequence analysis of bacterial cold shock proteins, we designed a protein (CspB-TB) that has the core residues of mesophilic protein from Bacillus subtilis(CspB-Bs) and altered distribution of surface charged residues. This designed protein was characterized by circular dichroism spectroscopy, and found to have secondary and tertiary structure similar to that of CspB-Bs. The activity of the CspB-TB protein as measured by the affinity to a single-stranded DNA (ssDNA) template at 25 degrees C is somewhat higher than that of CspB-Bs. Furthermore, the decrease in the apparent binding constant to ssDNA upon increase in temperature is much more pronounced for CspB-Bs than for CspB-TB. Temperature-induced unfolding (as monitored by differential scanning calorimetry and circular dichroism spectroscopy) and urea-induced unfolding experiments were used to compare the stabilities of CspB-Bs and CspB-TB. It was found that CspB-TB is approximately 20 degrees C more thermostable than CspB-Bs. The thermostabilization of CspB-TB relative to CspB-Bs is achieved by decrease in the enthalpy and entropy of unfolding without affecting their temperature dependencies, i.e. these proteins have similar heat capacity changes upon unfolding. These changes in the thermodynamic parameters result in the global stability function, i.e. Gibbs energy, deltaG(T), that is shifted to higher temperatures with only small changes in the maximum stability. Such a mechanism of thermostabilization, although predicted from the basic thermodynamic considerations, has never been identified experimentally.
authors.affiliation: Department of Biochemistry and Molecular Biology, Penn State University, College of Medicine, Hershey, PA 17033, USA. makhatadze@psu.edu
authors.forename: George I
authors.initials: GI
authors.lastname: Makhatadze
authors.forename: Vakhtang V
authors.initials: VV
authors.lastname: Loladze
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Gribenko
authors.forename: Maria M
authors.initials: MM
authors.lastname: Lopez
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D016415:Sequence Alignment; D055672:Static Electricity; D013499:Surface Properties; D013696:Temperature; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; C407094:cold shock protein, Bacillus