Shrake A, Ross P D
Division of Hematology, Food and Drug Administration, Bethesda, Maryland.
Biopolymers. 1992 Aug;32(8):925-40. doi: 10.1002/bip.360320804.
The presence of subsaturating levels of a high-affinity ligand has been demonstrated both by experiment and calculation to have far-reaching consequences on thermally induced protein denaturation due to the coupling between the protein denaturation and ligand-binding equilibria. Under such circumstances, a protein may undergo biphasic denaturation even though in the absence of ligand it exhibits a thermogram comprised of a single essentially symmetric endotherm. Up to now, the presence of just 2 maxima in the thermogram has been presented merely as an experimental observation or as the result of equilibrium computations. Here we develop a thermodynamic description of the linkage between these equilibria in which the number of cusps present in the thermogram correlates with the number of resolved steps in the plot of saturation level of remaining native protein vs temperature (i.e., the thermal binding curve). During thermally induced denaturation, the concentration of native protein decreases; thus, the native protein in effect is titrated with ligand at constant total ligand concentration. The free ligand concentration The free ligand concentration increases substantially through the release of bound ligand by unfolding protein thereby increasing the saturation level of the remaining native protein. The form of this thermal binding curve is a function of the number of ligand-binding sites on the protein, the magnitudes of the association constants, and the total ligand and total protein concentrations. As a result, the model predicts multiphasic denaturation of a single cooperative unit when the thermal binding curve consists of discrete multiple steps. The presence of only 2 maxima (i.e., a single cusp) in a thermogram for a protein with multiple sites on the native species derives from the form of the thermal binding curve, which in this case is a single-step sigmoidal plot, and not from the predominant denaturation of unliganded and fully liganded native species. In addition, it is shown that, in general, the contributions from the denaturation of individual native protein species are decidedly non-two-state in character; thus, simple deconvolution should not be carried out. The effects of nonzero values of delta Cp and d delta Cp/dT for denaturation and of changes in enthalpy and in heat capacity for ligand binding, as well as the interaction of ligand with the denatured protein, are explored also.
实验和计算均已证明，由于蛋白质变性与配体结合平衡之间的耦合作用，高亲和力配体的亚饱和水平的存在对热诱导的蛋白质变性具有深远影响。在这种情况下，即使在没有配体时蛋白质的热谱图由单个基本对称的吸热峰组成，蛋白质也可能经历双相变性。到目前为止，热谱图中仅出现2个最大值仅仅作为实验观察结果或平衡计算结果呈现出来。在此，我们建立了这些平衡之间联系的热力学描述，其中热谱图中尖点的数量与剩余天然蛋白质饱和度水平相对于温度的曲线（即热结合曲线）中解析出的步骤数量相关。在热诱导变性过程中，天然蛋白质的浓度降低；因此，实际上天然蛋白质是在总配体浓度恒定的情况下用配体进行滴定。通过未折叠蛋白质释放结合的配体，游离配体浓度大幅增加，从而提高了剩余天然蛋白质的饱和度水平。这种热结合曲线的形式是蛋白质上配体结合位点的数量、缔合常数的大小、总配体和总蛋白质浓度的函数。因此，当热结合曲线由离散的多个步骤组成时，该模型预测单个协同单元会发生多相变性。对于天然状态下具有多个位点的蛋白质，其热谱图中仅出现2个最大值（即单个尖点）源自热结合曲线的形式，在这种情况下是单步S形曲线，而不是来自未结合配体和完全结合配体的天然状态的主要变性。此外，研究表明，一般来说，单个天然蛋白质状态变性的贡献在性质上绝非二态性；因此，不应进行简单的去卷积操作。还探讨了变性时ΔCp和dΔCp/dT的非零值以及配体结合时焓和热容变化的影响，以及配体与变性蛋白质的相互作用。
