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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Probing osmolyte participation in the unfolding transition state of a protein
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Probing osmolyte participation in the unfolding transition state of a protein

机译：探索渗透质参与蛋白质的展开过渡状态

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摘要

Understanding the molecular mechanisms of osmolyte protection in protein stability has proved to be challenging. In particular, little is known about the role of osmolytes in the structure of the unfold ing transition state of a protein, the main determinant of its dynamics. We have developed an experimental protocol to directly probe the transition state of a protein in a range of osmolyte environments. We use an atomic force microscope in force-clamp mode to apply mechanical forces to the protein 127 and obtain force-dependent rate constants of protein unfolding. We measure the distance to the unfolding transition state. △x_u, along a 1D reaction coordinate imposed by mechanical force. We find that for the small osmolytes, ethylene glycol, propylene glycol, and glycerol, △x_u scales with the size of the molecule, whereas for lar ger osmolytes, sorbitol and sucrose, △x_u remains the same as that measured in water. These results are in agreement with steered molecular dynamics simulations that show that small osmolytes act as solvent bridges in the unfolding transition state structure, whereas only water molecules act as solvent bridges in large osmo lyte environments. These results demonstrate that novel force protocols combined with solvent substitution can directly probe angstrom changes in unfolding transition state structure. This approach creates new opportunities to gain molecular level under standing of the action of osmolytes in biomolecular processes.
机译：理解渗透蛋白保护蛋白稳定性的分子机制已被证明具有挑战性。特别地，关于渗透压在蛋白质的展开过渡状态的结构中的作用所知甚少，蛋白质是其动力学的主要决定因素。我们已经开发了一种实验方案，可以直接在一系列渗透压环境中探测蛋白质的过渡状态。我们使用力夹模式的原子力显微镜对蛋白质127施加机械力，并获得蛋白质展开的力相关速率常数。我们测量到展开过渡状态的距离。 △x_u，沿着由机械力施加的一维反作用坐标。我们发现，对于小的渗透液，乙二醇，丙二醇和甘油，△x_u随分子大小而缩放，而对于较大的渗透液，山梨糖醇和蔗糖，△x_u保持与在水中测得的相同。这些结果与指导分子动力学模拟相一致，该模拟表明，小渗透物在展开的过渡态结构中充当溶剂桥，而在大渗透物环境中只有水分子充当溶剂桥。这些结果表明，新颖的作用力方案与溶剂替代相结合可以直接探测展开转变状态结构中的埃变化。在渗透剂在生物分子过程中的作用下，这种方法创造了获得分子水平的新机会。

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