Pradeep Lovy, Udgaonkar Jayant B
National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore, India.
J Mol Biol. 2007 Feb 23;366(3):1016-28. doi: 10.1016/j.jmb.2006.11.064. Epub 2006 Dec 1.
To determine how the dynamics of the polypeptide chain in a protein molecule are coupled to the bulk solvent viscosity, the unfolding by urea of the small protein barstar was studied in the presence of two viscogens, xylose and glycerol. Thermodynamic studies of unfolding show that both viscogens stabilize barstar by a preferential hydration mechanism, and that viscogen and urea act independently on protein stability. Kinetic studies of unfolding show that while the rate-limiting conformational change during unfolding is dependent on the bulk solvent viscosity, eta, its rate does not show an inverse dependence on eta, as expected by Kramers' theory. Instead, the rate is found to be inversely proportional to an effective viscosity, eta + xi, where xi is an adjustable parameter which needs to be included in the rate equation. xi is found to have a value of -0.7 cP in xylose and -0.5 cP in glycerol, in the case of unfolding, at constant urea concentration as well as under isostability conditions. Hence, the unfolding protein chain does not experience the bulk solvent viscosity, but instead an effective solvent viscosity, which is lower than the bulk solvent viscosity by either 0.7 cP or 0.5 cP. A second important result is the validation of the isostability assumption, commonly used in protein folding studies but hitherto untested, according to which if a certain concentration of urea can nullify the effect of a certain concentration of viscogen on stability, then the same concentrations of urea and viscogen will also not perturb the free energy of activation of the unfolding of the protein.
为了确定蛋白质分子中多肽链的动力学如何与本体溶剂粘度相耦合，我们研究了在两种粘性剂木糖和甘油存在下，小蛋白质巴氏抑素（barstar）在尿素作用下的展开情况。展开过程的热力学研究表明，两种粘性剂均通过优先水合机制使巴氏抑素稳定，且粘性剂和尿素对蛋白质稳定性的作用相互独立。展开过程的动力学研究表明，虽然展开过程中限速构象变化取决于本体溶剂粘度η，但其速率并不像克莱默斯理论所预期的那样与η呈反比关系。相反，发现该速率与有效粘度η + ξ成反比，其中ξ是一个可调参数，需要包含在速率方程中。在尿素浓度恒定以及等稳定性条件下展开时，发现在木糖中ξ的值为 -0.7 cP，在甘油中为 -0.5 cP。因此，展开的蛋白质链所经历的不是本体溶剂粘度，而是一种有效溶剂粘度，它比本体溶剂粘度低0.7 cP或0.5 cP。第二个重要结果是验证了蛋白质折叠研究中常用但迄今未经测试的等稳定性假设，即如果一定浓度的尿素可以抵消一定浓度的粘性剂对稳定性的影响，那么相同浓度的尿素和粘性剂也不会干扰蛋白质展开的活化自由能。
