School of Life Sciences, Centre for Protein Science and Crystallography, The Chinese University of Hong Kong, Hong Kong, Shatin, Hong Kong SAR, China.
PLoS One. 2011;6(6):e21624. doi: 10.1371/journal.pone.0021624. Epub 2011 Jun 24.
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.
大多数嗜热蛋白往往具有更多的盐桥，通过提高和拓宽其蛋白质稳定性曲线来实现更高的热稳定性。虽然盐桥的稳定作用已经得到了广泛的研究，但关于盐桥如何影响蛋白质稳定性曲线的实验数据却很少。在这里，我们使用双突变体循环来确定对热稳定核糖体蛋白 L30e 中盐桥的成对相互作用能和 ΔC(p)贡献的温度依赖性。我们的结果表明，盐桥 E6/R92 和 E62/K46 的成对相互作用能是稳定的，并且对 298 到 348 K 的温度变化不敏感。另一方面，控制长程离子对 E90/R92 的成对相互作用能可以忽略不计。通过吉布斯-亥姆霍兹和基尔霍夫分析确定了所有单突变体和双突变体的 ΔC(p)。我们表明，这两个稳定的盐桥有助于降低 ΔC(p) 0.8-1.0 kJ mol⁻¹ K⁻¹。总之，我们的结果表明，嗜热蛋白中发现的额外盐桥通过降低 ΔC(p) 来提高蛋白质的热稳定性，从而提高蛋白质稳定性曲线的上移和变宽。
