Contribution of a buried hydrogen bond to lambda repressor folding kinetics.
Contribution of a buried hydrogen bond to lambda repressor folding kinetics.
复制标题
埋藏氢键对 lambda 阻遏物折叠动力学的贡献。
作者：
Myers,JK;Oas,TG
在单体λ阻遏物中，隐藏的残基Asp 14和Ser 77之间的氢键已通过将这些残基突变为丙氨酸而被去除。双突变体循环表明，相互作用使蛋白质的天然状态稳定了1.5 kcal/mol。去除的相互作用主要影响的展开速率，而不是折叠速率，这表明这种氢键基本上没有形成在蛋白质的折叠途径中的限速步骤。λ6-85的两个版本（野生型和更快折叠的G46 A/G48 A（WT*））中的突变显示出类似的效果。扩散-碰撞正确地预测了WT* 的行为，但不是野生型。我们的分析表明，螺旋3的折叠是沿着各种折叠途径的关键缓慢步骤，通常发生在14−77氢键形成之前。去除三级相互作用的实验，结合改变螺旋稳定性和扩散碰撞计算的实验，提供了一种解开小螺旋蛋白质折叠机制的策略。
A hydrogen bond between the buried residues Asp 14 and Ser 77 in monomeric lambda repressor has been removed by mutation of these residues to alanine. Double mutant cycles show that the interaction stabilizes the native state of the protein by 1.5 kcal/mol. Removal of the interaction affects mainly the unfolding rates and not the folding rates, suggesting that this hydrogen bond is not substantially formed in the rate-limiting steps in the folding pathways of the protein. Mutations in two versions of λ6-85, wild type and the faster folding G46A/G48A (WT*), show similar effects. Diffusion−collision correctly predicts the behavior of WT* but not of wild type. Our analysis suggests that folding of helix 3 is a crucial slow step along the various folding pathways and generally occurs before the formation of the 14−77 hydrogen bond. Experiments removing tertiary interactions, combined with experiments altering helical stability and diffusion−collision calculations, provide a strategy to unravel the folding mechanisms of small helical proteins.