Genetics, energetics and allostery during a billion years of hydrophobic protein core evolution

Protein folding is driven by the burial of hydrophobic amino acids in a tightly-packed core that excludes water. The genetics, biophysics and evolution of hydrophobic cores are not well understood, in part because of a lack of systematic experimental data on sequence combinations that do - and do not - constitute stable and functional cores. Here we randomize protein hydrophobic cores and evaluate their stability and function at scale. The data show that vast numbers of amino acid combinations can constitute stable protein cores but that these alternative cores frequently disrupt protein function because of allosteric effects. These strong allosteric effects are not due to complicated, highly epistatic fitness landscapes but rather, to the pervasive nature of allostery, with many individually small energy changes combining to disrupt function. Indeed both protein stability and ligand binding can be accurately predicted over very large evolutionary distances using additive energy models with a small contribution from pairwise energetic couplings. As a result, energy models trained on one protein can accurately predict core stability across hundreds of millions of years of protein evolution, with only rare energetic couplings that we experimentally identify limiting the transplantation of cores between highly diverged proteins. Our results reveal the simple energetic architecture of protein hydrophobic cores and suggest that allostery is a major constraint on sequence evolution.蛋白质折叠由疏水氨基酸（hydrophobic amino acids）埋入紧密堆积且排斥水分子的核心这一过程所驱动。目前，疏水核心（hydrophobic core）的遗传学、生物物理学与演化机制尚未得到充分阐明，部分原因在于缺乏针对可形成稳定功能性核心与无法形成该核心的氨基酸序列组合的系统性实验数据。本研究通过随机化改造蛋白质疏水核心，并大规模评估其稳定性与功能。实验数据表明，尽管大量氨基酸组合均可形成稳定的蛋白质核心，但这些替代型核心常因别构效应（allosteric effect）破坏蛋白质功能。这类显著的别构效应并非源于复杂的高度上位性适应度景观（epistatic fitness landscape），而是源于别构作用的普遍性——众多独立的微小能量变化共同累积，最终破坏蛋白质功能。事实上，借助仅包含少量成对能量耦合贡献的加性能量模型，我们可在极宽的演化跨度下精准预测蛋白质稳定性与配体结合能力。因此，基于单一蛋白质训练得到的能量模型，可在数亿年的蛋白质演化历程中精准预测核心稳定性；仅存在极少数经实验确认的成对能量耦合，会限制高度分化蛋白质之间的核心移植。本研究结果揭示了蛋白质疏水核心的简洁能量架构，并表明别构作用是制约序列演化的关键因素之一。