Quantitative measurements of protein-surface interaction thermodynamics.
Quantitative measurements of protein-surface interaction thermodynamics.
复制标题
蛋白质-表面相互作用热力学的定量测量。
DOI：
10.1073/pnas.1800287115
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发表时间：
2018
影响因子：
11.1
通讯作者：
Plaxco,KevinW
中科院分区：
文献类型：
--
作者：
Kurnik,Martin;Ortega,Gabriel;Dauphin-Ducharme,Philippe;Li,Hui;Caceres,Amanda;Plaxco,KevinW
虽然蛋白质在与生物表面相互作用时通常保持稳定，但它们经常在人造表面上展开并粘附。了解这种差异的物理化学起源将有助于开发基于蛋白质的传感器和其他技术，这些技术需要不损害蛋白质结构和功能的表面。然而，迄今为止，仅报道了少量此类人造表面，并且为什么这些表面支持功能性生物分子而其他表面则不支持的物理原理尚未确定。因此，我们开发了一种电化学方法来确定特定位点附着在化学明确的宏观表面上的蛋白质的折叠自由能。与本体溶液中看到的折叠自由能的比较提供了表面相互作用改变蛋白质稳定性程度的定量测量。作为原理验证，我们对 FynSH3 结构域进行了表征，该结构域位点特异性地附着在羟基涂覆的表面上。氯化胍变性后，蛋白质以可逆的二态方式展开，自由能与本体溶液中的自由能相差不超过 2 kJ/mol。假设排除的体积效应稳定了表面附着的蛋白质，这一观察结果表明存在与表面的相反的不稳定相互作用，在这些条件下，其大小相似。我们的技术构成了一种前所未有的实验工具，可以回答有关蛋白质与表面相互作用的分子尺度起源的长期存在的问题，并促进表面生物相容性的合理优化。
Whereas proteins generally remain stable upon interaction with biological surfaces, they frequently unfold on and adhere to artificial surfaces. Understanding the physicochemical origins of this discrepancy would facilitate development of protein-based sensors and other technologies that require surfaces that do not compromise protein structure and function. To date, however, only a small number of such artificial surfaces have been reported, and the physics of why these surfaces support functional biomolecules while others do not has not been established. Thus motivated, we have developed an electrochemical approach to determining the folding free energy of proteins site-specifically attached to chemically well-defined, macroscopic surfaces. Comparison with the folding free energies seen in bulk solution then provides a quantitative measure of the extent to which surface interactions alter protein stability. As proof-of-principle, we have characterized the FynSH3 domain site-specifically attached to a hydroxyl-coated surface. Upon guanidinium chloride denaturation, the protein unfolds in a reversible, two-state manner with a free energy within 2 kJ/mol of the value seen in bulk solution. Assuming that excluded volume effects stabilize surface-attached proteins, this observation suggests there are countervening destabilizing interactions with the surface that, under these conditions, are similar in magnitude. Our technique constitutes an unprecedented experimental tool with which to answer long-standing questions regarding the molecular-scale origins of protein−surface interactions and to facilitate rational optimization of surface biocompatibility.