Horovitz A, Fersht A R
MRC Unit for Protein Function and Design, University of Cambridge, U.K.
J Mol Biol. 1992 Apr 5;224(3):733-40. doi: 10.1016/0022-2836(92)90557-z.
The theory for measuring co-operativity between interactions in proteins by protein engineering experiments is developed by introducing a procedure for analysing increasing orders of synergy in a protein with increasing numbers of residues. The (pairwise) interaction energy (delta 2Gint) between two side-chains may be measured experimentally by a double-mutant cycle consisting of the wild-type protein, the two single mutants and the double mutant. This procedure may be extended to three residues to give a value for delta 3Gint for a triple-mutant cube, and to higher orders using multi-dimensional mutant space. We now show that delta 3Gint is the excess energy of adding all three chains compared with the sum of all the pairwise values of delta 2Gint for each of the constituent double-mutant cycles and the sum of all the single addition energies. This physical interpretation extends to higher orders of mutation. delta nGint (i.e. the interaction energy for n residues), thus, reveals the layers of synergy in interactions as a protein is built up. This procedure is applied to measuring changes in synergy during the refolding of barnase for the triad of salt-linked residues Asp8, Asp12 and Arg110, which are mutated to alanine residues. The value of delta 3Gint in the folded structure is 0.77(+/- 0.06) kcal mol-1 (i.e. the triad is 0.77 kcal mol-1 more stable than expected from the sum of the individual pairwise interactions and single contributions). The value of delta 3Gint is still significant in the transition state for unfolding (0.60(+/- 0.07) kcal mol-1) and in the folding intermediate (0.60(+/- 0.13 kcal mol-1)). These results show that synergistic interactions exist in barnase, in its transition state for unfolding and in a refolding intermediate. A direct measurement of the change of co-operativity between the folded state and the transition state for unfolding shows a decrease of 0.17(+/- 0.04) kcal mol-1, suggesting that the initial stages of protein unfolding may be accompanied by some loosening of structure in parts that still interact. The similar extent of co-operativity in the transition state for unfolding and the intermediate in refolding suggests that the intermediate is homogeneous, at least in the region of the salt-linked triad, as heterogeneity would lower the co-operativity.
通过引入一种分析随着蛋白质残基数量增加而协同作用增加顺序的程序，建立了一种通过蛋白质工程实验测量蛋白质中相互作用之间协同性的理论。两个侧链之间的（成对）相互作用能（δ2Gint）可以通过由野生型蛋白质、两个单突变体和双突变体组成的双突变循环进行实验测量。该程序可以扩展到三个残基，以得到三突变体立方体的δ3Gint值，并使用多维突变体空间扩展到更高阶。我们现在表明，δ3Gint是添加所有三条链的多余能量，与每个组成双突变循环的所有成对δ2Gint值之和以及所有单添加能量之和相比。这种物理解释扩展到更高阶的突变。因此，δnGint（即n个残基的相互作用能）揭示了随着蛋白质的构建，相互作用中协同作用的层次。该程序应用于测量在将盐连接的残基Asp8、Asp12和Arg110突变为丙氨酸残基时，巴那斯酶重折叠过程中协同性的变化。折叠结构中δ3Gint的值为0.77（±0.06）kcal mol-1（即三元组比单个成对相互作用和单个贡献之和预期的稳定性高0.77 kcal mol-1）。δ3Gint的值在解折叠的过渡态（0.60（±0.07）kcal mol-1）和折叠中间体（0.60（±0.13 kcal mol-1））中仍然显著。这些结果表明，巴那斯酶及其解折叠的过渡态和重折叠中间体中存在协同相互作用。对折叠态和解折叠过渡态之间协同性变化的直接测量显示降低了0.17（±0.04）kcal mol-1，这表明蛋白质解折叠的初始阶段可能伴随着仍相互作用部分结构的一些松动。解折叠过渡态和重折叠中间体中协同性的相似程度表明中间体是均匀的，至少在盐连接三元组区域是这样，因为异质性会降低协同性。
