title: Stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding.
abstract: Most thermophilic proteins tend to have more salt bridges, and achieve higher thermostability by up-shifting and broadening their protein stability curves. While the stabilizing effect of salt-bridge has been extensively studied, experimental data on how salt-bridge influences protein stability curves are scarce. Here, we used double mutant cycles to determine the temperature-dependency of the pair-wise interaction energy and the contribution of salt-bridges to ΔC(p) in a thermophilic ribosomal protein L30e. Our results showed that the pair-wise interaction energies for the salt-bridges E6/R92 and E62/K46 were stabilizing and insensitive to temperature changes from 298 to 348 K. On the other hand, the pair-wise interaction energies between the control long-range ion-pair of E90/R92 were negligible. The ΔC(p) of all single and double mutants were determined by Gibbs-Helmholtz and Kirchhoff analyses. We showed that the two stabilizing salt-bridges contributed to a reduction of ΔC(p) by 0.8-1.0 kJ mol⁻¹ K⁻¹. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing ΔC(p), leading to the up-shifting and broadening of the protein stability curves.
authors: School of Life Sciences, Centre for Protein Science and Crystallography, The Chinese University of Hong Kong, Hong Kong, Shatin, Hong Kong SAR, China.
authors: Chi-Ho
authors: CH
authors: Chan
authors: Tsz-Ha
authors: TH
authors: Yu
authors: Kam-Bo
authors: KB
authors: Wong
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D058767:Protein Unfolding; D012492:Salts / Q000737:chemistry*; D019712:Thermococcus / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D019843:Archaeal Proteins; D050505:Mutant Proteins; D012492:Salts