Horovitz A, Serrano L, Avron B, Bycroft M, Fersht A R
Department of Chemistry, University of Cambridge, U.K.
J Mol Biol. 1990 Dec 20;216(4):1031-44. doi: 10.1016/S0022-2836(99)80018-7.
Many of the interactions that stabilize proteins are co-operative and cannot be reduced to a sum of pairwise interactions. Such interactions may be analysed by protein engineering methods using multiple thermodynamic cycles comprising wild-type protein and all combinations of mutants in the interacting residues. There is a triad of charged residues on the surface of barnase, comprising residues Asp8, Asp12 and Arg110, that interact by forming two exposed salt bridges. The three residues have been mutated to alanine to give all the single, double and triple mutants. The free energies of unfolding of wild-type and the seven mutant proteins have been determined and the results analysed to give the contributions of the residues in the two salt bridges to protein stability. It is possible to isolate the energies of forming the salt bridges relative to the solvation of the separated ions by water. In the intact triad, the apparent contribution to the stabilization energy of the protein of the salt bridge between Asp12 and Arg110 is -1.25 kcal mol-1, whereas that of the salt bridge between Asp8 with Arg110 is -0.98 kcal mol-1. The strengths of the two salt bridges are coupled: the energy of each is reduced by 0.77 kcal mol-1 when the other is absent. The salt-linked triad, relative to alanine residues at the same positions, does not contribute to the stability of the protein since the favourable interactions of the salt bridges are more than offset by other electrostatic and non-electrostatic energy terms. Salt-linked triads occur in other proteins, for example, haemoglobin, where the energy of only the salt-bridge term is important and so the coupling of salt bridges could be of general importance to the stability and function of proteins.
许多稳定蛋白质的相互作用是协同性的，不能简化为成对相互作用的总和。此类相互作用可通过蛋白质工程方法进行分析，使用包含野生型蛋白质以及相互作用残基中所有突变体组合的多个热力学循环。核糖核酸酶 barnase 表面有一个由 Asp8、Asp12 和 Arg110 残基组成的带电三联体，它们通过形成两个暴露的盐桥相互作用。这三个残基已被突变为丙氨酸，从而得到所有单突变、双突变和三突变体。已测定野生型和七种突变体蛋白质的解折叠自由能，并对结果进行分析，以确定两个盐桥中残基对蛋白质稳定性的贡献。相对于分离离子被水溶剂化的情况，可以分离出形成盐桥的能量。在完整的三联体中，Asp12 与 Arg110 之间的盐桥对蛋白质稳定能的表观贡献为 -1.25 kcal mol-1，而 Asp8 与 Arg110 之间的盐桥为 -0.98 kcal mol-1。两个盐桥的强度是耦合的：当另一个盐桥不存在时，每个盐桥的能量会降低 0.77 kcal mol-1。相对于相同位置的丙氨酸残基，盐连接的三联体对蛋白质稳定性没有贡献，因为盐桥的有利相互作用被其他静电和非静电能量项更多地抵消了。盐连接的三联体存在于其他蛋白质中，例如血红蛋白，在那里仅盐桥项的能量很重要，因此盐桥的耦合可能对蛋白质的稳定性和功能具有普遍重要性。
