Schrank Travis P, Elam W Austin, Li Jing, Hilser Vincent J
Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.
Methods Enzymol. 2011;492:253-82. doi: 10.1016/B978-0-12-381268-1.00020-3.
Conformational fluctuations in proteins have emerged as an important aspect of biological function, having been linked to processes ranging from molecular recognition and catalysis to allostery and signal transduction. In spite of the realization of their importance, however, the connections between fluctuations and function have largely been empirical, even when they have been quantitative. Part of the problem in understanding the role of fluctuations in function is the fact that the mere existence of fluctuations complicates the interpretation of classic mutagenesis approaches. Namely, mutagenesis, which is typically targeted to an internal position (to elicit an effect), will change the fluctuations as well as the structure of the native state. Decoupling these effects is essential to an unambiguous understanding of the role of fluctuations in function. Here, we use a mutation strategy that targets surface-exposed sites in flexible parts of the molecule for mutation to glycine. Such mutations leave the ground-state structure unaffected. As a result, we can assess the nature of the fluctuations, develop a quantitative model relating fluctuations to function (in this case, molecular recognition), and unambiguously resolve the probabilities of the fluctuating states. We show that when this approach is applied to Escherichia coli adenylate kinase (AK), unique thermodynamic and structural insights are obtained, even when classic mutagenesis approaches targeted to the same region yield ambiguous results.
蛋白质的构象波动已成为生物功能的一个重要方面，与从分子识别、催化到变构和信号转导等一系列过程相关联。然而，尽管人们已经认识到其重要性，但波动与功能之间的联系在很大程度上仍是经验性的，即使是定量的联系也是如此。理解波动在功能中作用的部分问题在于，仅仅波动的存在就使经典诱变方法的解释变得复杂。也就是说，通常针对内部位置（以引发效应）的诱变会改变波动以及天然状态的结构。将这些效应解耦对于明确理解波动在功能中的作用至关重要。在这里，我们使用一种突变策略，将分子柔性部分表面暴露的位点突变为甘氨酸。这种突变不会影响基态结构。结果，我们可以评估波动的性质，建立一个将波动与功能（在这种情况下是分子识别）相关联的定量模型，并明确解析波动状态的概率。我们表明，当将这种方法应用于大肠杆菌腺苷酸激酶（AK）时，即使针对同一区域的经典诱变方法产生模糊结果，也能获得独特的热力学和结构见解。
