Main E R, Fulton K F, Jackson S E
Cambridge University Chemical Laboratory, U.K.
Biochemistry. 1998 Apr 28;37(17):6145-53. doi: 10.1021/bi973111s.
The context-dependent nature in which mutations affect protein stability was investigated using the FK506-binding protein, FKBP12. Thirty-four mutations were made at sites throughout the protein, including residues located in the hydrophobic core, the beta-sheet, and the solvent-exposed face of the alpha-helix. Urea-induced denaturation experiments were used to measure the change in stability of the mutants relative to that of the wild type (Delta DeltaGU-F). The results clearly show that the extent of destabilization, or stabilization, is highly context-dependent. Correlations were sought in order to link Delta DeltaGU-F to various structural parameters. The strongest correlation found was between Delta DeltaGU-F and N, the number of methyl(ene) groups within a 6 A radius of the group(s) deleted. For mutations of buried hydrophobic residues, a correlation coefficient of 0.73 (n = 16,where n is the number of points) was obtained. This increased to 0.81 (n = 24) on inclusion of mutations of partially buried hydrophobic residues. These data could be superimposed on data obtained for other proteins for which similarly detailed studies have been performed. Thus, the contribution to stability from hydrophobic side chains, independent of the extent to which a side chain is buried, can be estimated quantitatively using N. This correlation appears to be a general feature of all globular proteins. The effect on stability of mutating polar and charged residues in the alpha-helix and beta-sheet was also found to be highly context-dependent. Previous experimental and statistical studies have shown that specific side chains can stabilize the N-caps of alpha-helices in proteins. Substitutions of Ile56 to Thr and Asp at the N-cap of the alpha-helix of FKBP12, however, were found to be highly destabilizing. Thus, the intrinsic propensities of an amino acid for a particular element of secondary structure can easily be outweighed by tertiary packing factors. This study highlights the importance of packing density in determining the contribution of a residue to protein stability. This is the most important factor that should be taken into consideration in protein design.
利用FK506结合蛋白FKBP12研究了突变影响蛋白质稳定性的上下文依赖性。在整个蛋白质的位点上进行了34个突变，包括位于疏水核心、β折叠和α螺旋溶剂暴露面的残基。采用尿素诱导变性实验来测量突变体相对于野生型的稳定性变化（ΔΔGU-F）。结果清楚地表明，去稳定化或稳定化的程度高度依赖于上下文。为了将ΔΔGU-F与各种结构参数联系起来，进行了相关性分析。发现的最强相关性是在ΔΔGU-F与N之间，N是被删除基团6埃半径内亚甲基的数量。对于埋藏的疏水残基突变，获得的相关系数为0.73（n = 16，其中n是点数）。当纳入部分埋藏的疏水残基突变时，这一系数增加到0.81（n = 24）。这些数据可以叠加到对其他蛋白质进行过类似详细研究的数据上。因此，可以使用N定量估计疏水侧链对稳定性的贡献，而与侧链埋藏程度无关。这种相关性似乎是所有球状蛋白质的一个普遍特征。还发现，α螺旋和β折叠中极性和带电残基的突变对稳定性的影响也高度依赖于上下文。先前的实验和统计研究表明，特定的侧链可以稳定蛋白质中α螺旋的N端帽。然而，发现FKBP12的α螺旋N端帽处的Ile56被Thr和Asp取代时具有高度的去稳定性。因此，氨基酸对特定二级结构元件的内在倾向很容易被三级包装因素所抵消。这项研究强调了堆积密度在确定残基对蛋白质稳定性贡献方面的重要性。这是蛋白质设计中应考虑的最重要因素。
