Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois 61801, USA.
J Chem Phys. 2011 Jul 7;135(1):015102. doi: 10.1063/1.3607605.
Protein stability is measured by denaturation: When solvent conditions are changed (e.g., temperature, denaturant concentration, or pH) the protein population switches between thermodynamic states. The resulting denaturation curves have baselines. If the baselines are steep, nonlinear, or incomplete, it becomes difficult to characterize protein denaturation. Baselines arise because the chromophore probing denaturation is sensitive to solvent conditions, or because the thermodynamic states evolve structurally when solvent conditions are changed, or because the barriers are very low (downhill folding). Kinetics can largely eliminate such baselines: Relaxation of chromophores, or within thermodynamic states, is much faster than the transition over activation barriers separating states. This separation of time scales disentangles population switching between states (desired signal) from chromophore or population relaxation within states (baselines). We derive simple formulas to extract unfolding thermodynamics from kinetics. The formulas are tested with model data and with a difficult experimental test case: the apparent two-state folder PI3K SH3 domain. Its melting temperature T(m) can be extracted reliably by our "thermodynamics from kinetics approach," even when conventional fitting is unreliable.
当溶剂条件发生变化（例如温度、变性剂浓度或 pH 值）时，蛋白质群体在热力学状态之间切换。由此产生的变性曲线有基线。如果基线陡峭、非线性或不完整，则难以描述蛋白质变性。基线的出现是因为发色团探测变性对溶剂条件敏感，或者因为在改变溶剂条件时热力学状态在结构上演变，或者因为势垒非常低（下坡折叠）。动力学可以在很大程度上消除这些基线：发色团的弛豫，或在热力学状态内，比跨越状态之间的激活势垒的转变快得多。这种时间尺度的分离将状态之间的种群转换（所需信号）与状态内的发色团或种群弛豫（基线）区分开来。我们推导出了从动力学中提取展开热力学的简单公式。这些公式通过模型数据和一个困难的实验测试案例进行了测试：表观两态文件夹 PI3K SH3 结构域。即使常规拟合不可靠，我们的“从动力学中提取热力学”方法也可以可靠地提取其熔点 T(m)。
