Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark.
J Am Chem Soc. 2011 Jan 19;133(2):271-8. doi: 10.1021/ja105388k. Epub 2010 Dec 17.
The unusual stability of a structured but locally flexible protein, human growth hormone (hGH) at pH 2.7, was investigated using the temperature dependence of the nanosecond-picosecond dynamics of the backbone amide groups obtained from (15)N NMR relaxation data. It is found that the flexibility of the backbone of the helices decreases with temperature in the range from 24 °C to ∼40 °C, corresponding to an increasing stability. A concomitant increase with temperature of the electrostatic interactions between charged residues forming an interhelical network of salt bridges at the center of the four-helix core suggests that these interactions give rise to the decreasing flexibility and increasing stability of the protein. However, numerous hydrophobic interactions in the interior of the four-helix core may also contribute. Above ∼40 °C, where the thermal energy overcomes the electrostatic and hydrophobic interactions, a substantial increase in the flexibility of the helix backbones results in a highly positive contribution from the local conformational heat capacity, C(p, conf), of the helix backbones to the total heat capacity, C(p), of the protein. This reduces the change in heat capacity upon unfolding, ΔC(p), increases the change in the Gibbs free energy, ΔG(unfold), and stabilizes the protein at high temperatures. A similar decrease in flexibility is found near other salt bridges in hGH and in Calmodulin and may be of general importance for the thermostability of proteins and, in particular, of the salt bridge intensive thermophilic proteins.
在 pH 值为 2.7 时，结构稳定但局部灵活的蛋白质——人生长激素（hGH）的异常稳定性，使用从（15）N NMR 弛豫数据获得的酰胺基回波峰的纳秒-皮秒动力学的温度依赖性进行了研究。研究发现，在 24°C 到约 40°C 的温度范围内，螺旋的骨架柔韧性随温度降低，这表明稳定性增加。带电荷残基之间的静电相互作用与温度呈正相关，这些残基在四螺旋核心的中心形成了一个螺旋间的盐桥网络，这表明这些相互作用导致了蛋白质柔韧性的降低和稳定性的增加。然而，在四螺旋核心内部存在许多疏水性相互作用也可能起到作用。在高于约 40°C 的温度下，热能克服了静电和疏水性相互作用，螺旋骨架的柔韧性显著增加，导致螺旋骨架的局部构象热容 C(p, conf)对蛋白质总热容 C(p)的贡献呈高度正贡献。这降低了折叠过程中热容的变化量 ΔC(p)，增加了吉布斯自由能的变化量 ΔG(unfold)，并使蛋白质在高温下稳定。在 hGH 和钙调蛋白中的其他盐桥附近也发现了类似的柔韧性降低，这可能对蛋白质的热稳定性，特别是对盐桥密集的嗜热蛋白质的热稳定性具有普遍重要性。
