Investigating Homology between Proteins using Energetic Profiles

Accumulated experimental observations demonstrate that protein stability is often preserved upon conservative point mutation. In contrast, less is known about the effects of large sequence or structure changes on the stability of a particular fold. Almost completely unknown is the degree to which stability of different regions of a protein is generally preserved throughout evolution. In this work, these questions are addressed through thermodynamic analysis of a large representative sample of protein fold space based on remote, yet accepted, homology. More than 3,000 proteins were computationally analyzed using the structural-thermodynamic algorithm COREX/BEST. Estimated position-specific stability (i.e., local Gibbs free energy of folding) and its component enthalpy and entropy were quantitatively compared between all proteins in the sample according to all-vs.-all pairwise structural alignment. It was discovered that the local stabilities of homologous pairs were significantly more correlated than those of non-homologous pairs, indicating that local stability was indeed generally conserved throughout evolution. However, the position-specific enthalpy and entropy underlying stability were less correlated, suggesting that the overall regional stability of a protein was more important than the thermodynamic mechanism utilized to achieve that stability. Finally, two different types of statistically exceptional evolutionary structure-thermodynamic relationships were noted. First, many homologous proteins contained regions of similar thermodynamics despite localized structure change, suggesting a thermodynamic mechanism enabling evolutionary fold change. Second, some homologous proteins with extremely similar structures nonetheless exhibited different local stabilities, a phenomenon previously observed experimentally in this laboratory. These two observations, in conjunction with the principal conclusion that homologous proteins generally conserved local stability, may provide guidance for a future thermodynamically informed classification of protein homology.

累积的实验观测表明，保守点突变（conservative point mutation）通常可维持蛋白质的稳定性。与之相对，学界对较大序列或结构变化对特定蛋白质折叠（fold）稳定性的影响仍知之甚少。几乎完全未知的是，在整个进化历程中，蛋白质不同区域的稳定性究竟能在多大程度上得到普遍保留。本研究针对蛋白质折叠空间的大型代表性样本，基于远程且经公认的同源性（homology）开展热力学分析，以此解答上述问题。研究团队借助结构热力学算法COREX/BEST，对超过3000种蛋白质进行了计算分析。通过全对全两两结构比对（all-vs.-all pairwise structural alignment），研究人员对样本中所有蛋白质的位点特异性稳定性估计值（即局部折叠吉布斯自由能（Gibbs free energy of folding））及其组成要素焓（enthalpy）和熵（entropy）进行了定量比较。研究发现，同源蛋白对的局部稳定性相关性显著高于非同源蛋白对，这表明局部稳定性在进化过程中确实普遍得到了保守保留。不过，支撑稳定性的位点特异性焓与熵的相关性却较低，这提示蛋白质的整体区域稳定性，相较于实现该稳定性所采用的热力学机制，更为重要。最后，本研究观测到两类统计学上异常的进化-结构热力学关联：其一，诸多同源蛋白即便存在局部结构变化，仍具备相似的热力学特征，这暗示存在可介导进化性折叠改变的热力学机制；其二，部分同源蛋白尽管结构高度相似，却呈现出不同的局部稳定性——这一现象此前已在本实验室的实验中被观测到。上述两项观测结果，结合“同源蛋白普遍保留局部稳定性”这一核心结论，可为未来基于热力学信息的蛋白质同源性分类研究提供指导。