Choe S E, Li L, Matsudaira P T, Wagner G, Shakhnovich E I
Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
J Mol Biol. 2000 Nov 17;304(1):99-115. doi: 10.1006/jmbi.2000.4190.
We report the distribution of hydrophobic core contacts during the folding reaction transition state for villin 14T, a small 126-residue protein domain. The solution structure of villin 14T contains a central beta-sheet with two flanking hydrophobic cores; transition states for this protein topology have not been previously studied. Villin 14T has no disulfide bonds or cis-proline residues in its native state; it folds reversibly, and in an apparently two-state manner under some conditions. To map the hydrophobic core contacts in the transition state, 27 point mutations were generated at positions spread throughout the two hydrophobic cores. After each point mutation, comparison of the change in folding kinetics with the equilibrium destabilization indicates whether the site of mutation is stabilized in the transition state. The results show that the folding nucleus, or the sub-region with the strongest transition state contacts, is located in one of the two hydrophobic cores (the predominantly aliphatic core). The other hydrophobic core, which is mostly aromatic, makes much weaker contacts in the transition state. This work is the first transition state mapping for a protein with multiple major hydrophobic cores in a single folding unit; the hydrophobic cores cannot be separated into individual folding subdomains. The stabilization of only one hydrophobic core in the transition state illustrates that hydrophobic core formation is not intrinsically capable of nucleating folding, but must also involve the right specific interactions or topological factors in order to be kinetically important.
我们报告了小的126个残基的肌动蛋白结合蛋白14T（villin 14T）折叠反应过渡态期间疏水核心接触的分布情况。villin 14T的溶液结构包含一个中央β-折叠片层以及两个侧翼疏水核心；此前尚未研究过这种蛋白质拓扑结构的过渡态。villin 14T在其天然状态下没有二硫键或顺式脯氨酸残基；它可逆折叠，并且在某些条件下以明显的两态方式折叠。为了绘制过渡态中的疏水核心接触图，在遍布两个疏水核心的位置产生了27个点突变。每个点突变后，将折叠动力学的变化与平衡失稳进行比较，以表明突变位点在过渡态是否稳定。结果表明，折叠核，即具有最强过渡态接触的子区域，位于两个疏水核心之一（主要是脂肪族核心）中。另一个主要是芳香族的疏水核心在过渡态中的接触要弱得多。这项工作是对单个折叠单元中具有多个主要疏水核心的蛋白质进行的首次过渡态图谱绘制；这些疏水核心不能被分离成单独的折叠亚结构域。过渡态中只有一个疏水核心的稳定表明，疏水核心的形成本身并不能引发折叠，还必须涉及正确的特定相互作用或拓扑因素才能在动力学上具有重要意义。
