COOPERATIVE INTERACTIONS DURING PROTEIN FOLDING
COOPERATIVE INTERACTIONS DURING PROTEIN FOLDING
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DOI：
10.1016/0022-2836(92)90557-z
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发表时间：
1992-04-05
影响因子：
5.6
通讯作者：
FERSHT, AR
中科院分区：
文献类型：
--
作者：
HOROVITZ, A;FERSHT, AR
通过蛋白质工程实验测量蛋白质相互作用之间的协同性的理论是通过引入用于分析随着残基数目的增加蛋白质中的协同性的增加的顺序的程序来开发的。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体循环实验测量。该程序可以扩展到三个残基以给出三突变体立方体的Δ 3Gint的值，并且可以使用多维突变体空间扩展到更高阶。我们现在证明，Δ 3Gint是所有三条链相加的多余能量，与每个组成双突变循环的所有成对值（Δ2Gint）之和以及所有单次相加能量之和相比。这种物理解释延伸到更高阶的突变。因此，ΔnGint（即n个残基的相互作用能）揭示了蛋白质构建时相互作用中的协同作用层。此程序适用于测量的协同作用的变化，在重新折叠的芽孢杆菌RNA酶的三联体的盐连接的残基Asp8，Asp12和Arg110，这是突变为丙氨酸残基。折叠结构中Δ 3Gin的值为0.77（±0.06）kcal mol−1（即三联体比单个成对相互作用和单个贡献的总和稳定0.77 kcal mol− 1）。Δ 3Gin的值在去折叠的过渡态（0.60（±0.07）kcal mol−1）和折叠的中间态（0.60（±0.13 kcal mol−1））中仍然是显著的。这些结果表明，协同相互作用存在于芽孢杆菌RNA酶，在其去折叠的过渡态和在复性中间体。直接测量折叠态和去折叠过渡态之间的协同性变化，显示出0.17（±0.04）kcal mol−1的下降，这表明蛋白质去折叠的初始阶段可能伴随着仍然相互作用的部分的结构松动。在展开的过渡态和重折叠的中间体中的相似程度的协同性表明，中间体是均匀的，至少在盐连接的三联体的区域中，因为异质性会降低协同性。
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 (Δ2Gint) 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 Δ3Gintfor a triple-mutant cube, and to higher orders using multi-dimensional mutant space. We now show that Δ3Gintis the excess energy of adding all three chains compared with the sum of all the pairwise values of (Δ2Gint) 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. ΔnGint(i.e. the interaction energy fornresidues), 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 Δ3Gintin the folded structure is 0.77(±0.06) kcal mol−1(i.e. the triad is 0.77 kcal mol−1more stable than expected from the sum of the individual pairwise interactions and single contributions). The value of Δ3Gintis 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.