Brun Ludovic, Isom Daniel G, Velu Priya, García-Moreno Bertrand, Royer Catherine Ann
INSERM U554, Montpellier, F-34090 France.
Biochemistry. 2006 Mar 21;45(11):3473-80. doi: 10.1021/bi052638z.
A complete description of the mechanisms of protein folding requires knowledge of the structural and physical character of the folding transition state ensembles (TSEs). A key question concerning the role of hydration of the hydrophobic core in determining folding mechanisms remains. To address this, we probed the state of hydration of the TSE of staphylococcal nuclease (SNase) by examining the fluorescence-detected pressure-jump relaxation behavior of six SNase variants in which a residue in the hydrophobic core, Val-66, was replaced with polar or ionizable residues (Lys, Arg, His, Asp, Glu, and Asn). Because of a large positive activation volume for folding, the major effect of pressure on the wild-type protein is to decrease the folding rate. By the time wild-type SNase reaches the folding transition state, most water has already been expelled from its hydrophobic core. In contrast, the major effect of pressure on the variant proteins is an increase in the unfolding rate due to a large negative activation volume for unfolding. This results from a significant increase in the level of hydration of the TSE when an internal ionizable group is present. These data confirm that the role of water in the folding reaction can differ from protein to protein and that even a single substitution in a critical position can modulate significantly the properties of the TSE.
蛋白质折叠机制的完整描述需要了解折叠过渡态系综（TSEs）的结构和物理特性。关于疏水核心的水合作用在确定折叠机制中的作用这一关键问题仍然存在。为了解决这个问题，我们通过检测六种葡萄球菌核酸酶（SNase）变体的荧光检测压力跳跃弛豫行为，探究了SNase的TSE的水合状态。在这些变体中，疏水核心中的一个残基Val-66被极性或可电离残基（赖氨酸、精氨酸、组氨酸、天冬氨酸、谷氨酸和天冬酰胺）取代。由于折叠具有较大的正活化体积，压力对野生型蛋白质的主要影响是降低折叠速率。当野生型SNase达到折叠过渡态时，其疏水核心中的大部分水已经被排出。相比之下，压力对变体蛋白质的主要影响是由于去折叠具有较大的负活化体积而导致去折叠速率增加。这是由于当存在内部可电离基团时，TSE的水合水平显著增加所致。这些数据证实，水在折叠反应中的作用可能因蛋白质而异，而且即使在关键位置进行单个取代也能显著调节TSE的性质。
