Cosolvent Exclusion Drives Protein Stability in Trimethylamine <i>N</i>‑Oxide and Betaine Solutions

Using a combination of molecular dynamics simulation, dialysis experiments, and electronic circular dichroism measurements, we studied the solvation thermodynamics of proteins in two osmolyte solutions, trimethylamine N-oxide (TMAO) and betaine. We showed that existing force fields are unable to capture the solvation properties of the proteins lysozyme and ribonuclease T1 and that the inaccurate parametrization of protein–osmolyte interactions in these force fields promoted an unphysical strong thermal denaturation of the trpcage protein. We developed a novel force field for betaine (the KBB force field) which reproduces the experimental solution Kirkwood–Buff integrals and density. We further introduced appropriate scaling to protein–osmolyte interactions in both the betaine and TMAO force fields which led to successful reproduction of experimental protein–osmolyte preferential binding coefficients for lysozyme and ribonuclease T1 and prevention of the unphysical denaturation of trpcage in osmolyte solutions. Correct parametrization of protein–TMAO interactions also led to the stabilization of the collapsed conformations of a disordered elastin-like peptide, while the uncorrected parameters destabilized the collapsed structures. Our results establish that the thermodynamic stability of proteins in both betaine and TMAO solutions is governed by osmolyte exclusion from proteins.我们结合分子动力学模拟、透析实验与电子圆二色谱测量，对三甲基胺N-氧化物（trimethylamine N-oxide, TMAO）和甜菜碱（betaine）两种渗透剂溶液中的蛋白质溶剂化热力学展开了研究。研究表明，现有分子力场无法准确刻画溶菌酶与核糖核酸酶T1的溶剂化性质；这类力场中蛋白质-渗透剂相互作用的参数化失当，会导致trpcage蛋白出现非物理性的剧烈热变性。我们开发了一种针对甜菜碱的新型力场（KBB力场），该力场能够复现实验测得的溶液柯克伍德-布鲁夫积分（Kirkwood–Buff integrals）与溶液密度。我们进一步对甜菜碱和TMAO力场中的蛋白质-渗透剂相互作用进行了合理的缩放校正，成功复现了溶菌酶与核糖核酸酶T1的实验蛋白质-渗透剂优先结合系数，并避免了trpcage蛋白在渗透剂溶液中发生非物理性变性。对蛋白质-TMAO相互作用的正确参数化，还可稳定无序弹性蛋白样肽的塌缩构象，而未校正的参数则会瓦解此类塌缩结构。本研究证实，甜菜碱与TMAO溶液中蛋白质的热力学稳定性，均由渗透剂从蛋白质表面的排阻效应所主导。