stabilizing salt-bridge enhances protein thermostability by reducing the heat capacity change of unfolding稳定盐桥提高蛋白质热稳定性降低的热容变化展开.pdfVIP

Stabilizing Salt-Bridge Enhances Protein Thermostability by Reducing the Heat Capacity Change of Unfolding Chi-Ho Chan, Tsz-Ha Yu, Kam-Bo Wong* School of Life Sciences, Centre for Protein Science and Crystallography, The Chinese University of Hong Kong, Hong Kong, Shatin, Hong Kong SAR, China 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 DCp 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 DCp 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 DCp by 0.8–1.0 kJ mol21 K21. Taken together, our results suggest that the extra salt-bridges found in thermophilic proteins enhance the thermostability of proteins by reducing DCp, leading to the up-shifting and broadening of the protein stability curves. Citation: Chan C-H, Yu T-H, Wong K-B (2011) Stabilizing Salt-Bridge Enhances Protein Thermostability by Reducing the Heat Capacity Change of Unfolding. PLoS ONE 6(6): e21624. doi:10.1371/journal.pone.0021624 Editor: Annalisa Pastore, National Institute for Medical Research