Matthews J M, Fersht A R
MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, University Chemical Laboratory, U.K.
Biochemistry. 1995 May 23;34(20):6805-14. doi: 10.1021/bi00020a027.
The structure of alpha-helix 1 (residues 6-18) in the transition state for the unfolding of barnase has been previously characterized by comparing the kinetics and thermodynamics of folding of wild-type protein with those of mutants whose side chains have been cut back, in the main, to that of alanine. The structure of the transition state has now been explored further by comparing the kinetics and thermodynamics of folding of glycine mutants with those of the alanine mutants at solvent-exposed positions in the alpha-helices of barnase. Such "Ala-->Gly scanning" provides a general procedure for examining the structure of solvent-exposed regions in the transition state. A gradual change of structure of the transition state was detected as helix 1 becomes increasingly destabilized on mutation. The extent of change of structure of helix 1 in the transition state for the mutant proteins was probed by a further round of Ala-->Gly scanning of those mutants. Destabilization of the helix 1 was found to cause the overall transition state for unfolding to become closer in structure to that of the folded protein. This is analogous to the conventional Hammond effect in physical-organic chemistry whereby the transition state moves parallel to the reaction coordinate with change in structure. But, paradoxically, the structure of helix 1 itself becomes less folded in the transition state as helix 1 becomes destabilized. This is analogous, however, to the rarer anti-Hammond effect in which there is movement perpendicular to the reaction coordinate. These observations are rationalized by plotting correlation diagrams of degree of formation of individual elements of structure against the degree of formation of overall structure in the transition state. There is a relatively smooth movement of the degree of compactness in the transition state against changes in activation energy on mutation that suggests a smooth movement of the transition state along the energy surface on mutation rather than a switch between two different parallel pathways. The results are consistent with the transition state having closely spaced energy levels. Helix 1, which appears to be an initiation point and forms early in the folding of wild-type protein, may be radically destabilized to the extent that it forms late in the folding of mutants. The order of events in folding may thus not be crucial.
之前，通过比较野生型蛋白与侧链大多被截短为丙氨酸的突变体的折叠动力学和热力学，对核糖核酸酶 barnase 展开的过渡态中α-螺旋 1（第 6 - 18 位残基）的结构进行了表征。现在，通过比较甘氨酸突变体与 barnase 的α-螺旋中溶剂暴露位置的丙氨酸突变体的折叠动力学和热力学，进一步探究了过渡态的结构。这种“Ala→Gly 扫描”为研究过渡态中溶剂暴露区域的结构提供了一种通用方法。随着螺旋 1 在突变时变得越来越不稳定，检测到过渡态结构的逐渐变化。通过对这些突变体进行新一轮的 Ala→Gly 扫描，探究了突变体蛋白在过渡态中螺旋 1 结构的变化程度。发现螺旋 1 的不稳定会导致展开的整体过渡态在结构上更接近折叠蛋白的结构。这类似于物理有机化学中的传统哈蒙德效应，即过渡态随着结构变化平行于反应坐标移动。但矛盾的是，随着螺旋 1 变得不稳定，螺旋 1 本身在过渡态中的折叠程度会降低。然而，这类似于罕见的反哈蒙德效应，即存在垂直于反应坐标的移动。通过绘制过渡态中单个结构元素的形成程度与整体结构形成程度的相关图，对这些观察结果进行了合理化解释。过渡态中的紧凑程度随着突变时活化能的变化有相对平滑的移动，这表明过渡态在突变时沿着能量表面平滑移动，而不是在两条不同的平行途径之间切换。结果与过渡态具有紧密间隔的能级一致。螺旋 1 似乎是一个起始点，在野生型蛋白折叠早期形成，在突变体折叠后期可能会被极大地 destabilized。因此，折叠过程中事件的顺序可能并不关键。
