Schmittschmitt Jason P, Scholtz J Martin
Department of Medical Biochemistry and Genetics, Center for Advanced Biomolecular Research, Texas A&M University, College Station, Texas 77843-1114, USA.
Biochemistry. 2004 Feb 10;43(5):1360-8. doi: 10.1021/bi0357412.
Many small, single-domain proteins show equilibrium and kinetic folding mechanisms that appear to be adequately described as two state. The two-state model makes several predictions that can be tested experimentally. First, the conformational stability determined at or extrapolated to a set of reference conditions should be independent of the measurement method (thermal or solvent denaturation or hydrogen exchange). Second, model-independent measures of the cardinal thermodynamic parameters (T(m), DeltaH) as determined from direct calorimetric means should be identical to those determined from the two-state analysis of thermal unfolding data. Third, the ratio of the kinetic folding and unfolding rate constants should be equal to K(eq) determined from an equilibrium measurement under the same conditions. Here, we show that the wild-type HPr protein from Bacillus subtilis does not meet all of these criteria under our standard conditions. However, if we replace the side chain of Asp69, or add moderate concentrations of salt, we find excellent two-state behavior in both equilibrium and kinetic folding. Thus, for this protein and possibly others, very subtle changes in the primary structure or in the solution conditions can dramatically alter the relative stabilities of the native intermediate, and unfolded ensembles can cause an observable change in the nature of the folding mechanism.
许多小的单结构域蛋白表现出的平衡和动力学折叠机制似乎可以充分地描述为两态。两态模型做出了几个可以通过实验进行检验的预测。首先，在一组参考条件下测定或外推得到的构象稳定性应与测量方法（热变性或溶剂变性或氢交换）无关。其次，通过直接量热法确定的基本热力学参数（T(m)，ΔH）的与模型无关的测量值应与通过热变性数据的两态分析确定的值相同。第三，动力学折叠和去折叠速率常数的比值应等于在相同条件下通过平衡测量确定的K(eq)。在此，我们表明，在我们的标准条件下，来自枯草芽孢杆菌的野生型HPr蛋白并不满足所有这些标准。然而，如果我们替换Asp69的侧链，或添加适量浓度的盐，我们发现在平衡和动力学折叠中都有出色的两态行为。因此,对于这种蛋白以及可能的其他蛋白，一级结构或溶液条件中非常细微的变化都可能显著改变天然中间体和未折叠集合体的相对稳定性，从而导致折叠机制的性质发生可观察到的变化。
