Kuttner Yosef Yehuda, Kozer Noga, Segal Eugenia, Schreiber Gideon, Haran Gilad
Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel.
J Am Chem Soc. 2005 Nov 2;127(43):15138-44. doi: 10.1021/ja053681c.
The association of two proteins is preceded by a mutual diffusional search in solution. The role of translational and rotational diffusion in this process has been studied theoretically for many years. However, systematic experimental verification of theoretical results is still lacking. We report here measurements of association rates of the proteins beta-lactamase (TEM) and beta-lactamase inhibitor protein (BLIP) in solutions of glycerol and poly(ethylene glycol) of increasing viscosity. We also measured translational and rotational diffusion in the same solutions, using fluorescence correlation spectroscopy and fluorescence anisotropy, respectively. It is found that in glycerol both translational and rotational diffusion rates are inversely dependent on viscosity, as predicted by the classical Stokes-Einstein relations, while the association rate depends nonlinearly on viscosity. In contrast, the association rate depends only weakly on the viscosity of the polymer solutions, which results in a similar weak dependence of k(on) on viscosity. The data are modeled using the theory of diffusion-limited association. Deviations from the theory are explained by a short-range solute-induced repulsion between the proteins in glycerol solution and an attractive depletion interaction generated by the polymers. These results open the way to the creation of a unified framework for all nonspecific effects involved in the protein association process, as well as to better theoretical understanding of these effects. Further, they reflect on the complex factors controlling protein association within the crowded environment of cells and suggest that a high concentration of macromolecules does not significantly impede protein association.
两种蛋白质的结合之前是在溶液中的相互扩散搜索。平移扩散和旋转扩散在这个过程中的作用已经在理论上研究了很多年。然而，理论结果的系统实验验证仍然缺乏。我们在此报告在甘油和聚乙二醇溶液中，随着粘度增加，β-内酰胺酶（TEM）和β-内酰胺酶抑制剂蛋白（BLIP）的结合速率的测量结果。我们还分别使用荧光相关光谱和荧光各向异性测量了相同溶液中的平移扩散和旋转扩散。结果发现，在甘油中，平移扩散速率和旋转扩散速率都如经典的斯托克斯 - 爱因斯坦关系所预测的那样，与粘度成反比，而结合速率则非线性地依赖于粘度。相比之下，结合速率仅微弱地依赖于聚合物溶液的粘度，这导致k（on）对粘度的依赖也较弱。使用扩散限制结合理论对数据进行建模。甘油溶液中蛋白质之间的短程溶质诱导排斥以及聚合物产生的吸引性耗尽相互作用解释了与该理论的偏差。这些结果为建立蛋白质结合过程中所有非特异性效应的统一框架开辟了道路，也有助于对这些效应有更好的理论理解。此外，它们反映了细胞拥挤环境中控制蛋白质结合的复杂因素，并表明高浓度的大分子不会显著阻碍蛋白质结合。
