Vaughan Cara K, Harryson Pia, Buckle Ashley M, Fersht Alan R
Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, England.
Acta Crystallogr D Biol Crystallogr. 2002 Apr;58(Pt 4):591-600. doi: 10.1107/s0907444902001567. Epub 2002 Mar 22.
Double-mutant cycles are widely used in the field of protein engineering to measure intermolecular and intramolecular interactions. Ideally, there should be no structural rearrangement of the protein on making the two single mutations and the double mutation within the cycle. However, structural pertubation on mutation does not preclude the use of this method, providing the sum of the changes in the single mutants equals the change in the double mutant. In this way, the energy associated with any structural rearrangement cancels in the double-mutant cycle. Previously, the contribution of a buried salt bridge between Arg69 and Asp93 in barnase to the stability of the folded protein has been determined by double-mutant cycle analysis. In order to determine whether the measured interaction of -14.0 kJ mol(-1) represents the true interaction energy, the crystal structure of each mutant within the double-mutant cycle was solved. Although mutation results in structural shifts, the majority of those in the single mutants are also found in the double mutant; their energetic effects in the double-mutant cycle are therefore cancelled. This study highlights the robust nature of the double-mutant cycle analysis.
双突变循环在蛋白质工程领域被广泛用于测量分子间和分子内的相互作用。理想情况下，在进行循环中的两个单突变和双突变时，蛋白质不应发生结构重排。然而，突变引起的结构扰动并不妨碍该方法的使用，只要单突变体变化的总和等于双突变体的变化即可。通过这种方式，与任何结构重排相关的能量在双突变循环中相互抵消。此前，通过双突变循环分析已经确定了核糖核酸酶 barnase 中 Arg69 和 Asp93 之间一个埋藏盐桥对折叠蛋白稳定性的贡献。为了确定测得的 -14.0 kJ mol⁻¹ 的相互作用是否代表真实的相互作用能量，解析了双突变循环中每个突变体的晶体结构。尽管突变会导致结构变化，但单突变体中的大多数变化也出现在双突变体中；因此它们在双突变循环中的能量效应相互抵消。这项研究突出了双突变循环分析的稳健性。
