Department of Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
Proteins. 2012 Mar;80(3):858-70. doi: 10.1002/prot.23243. Epub 2011 Dec 13.
Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.
优化天然状态下表面暴露的电荷-电荷相互作用已成为增强蛋白质稳定性的有效手段；但静电相互作用对蛋白质折叠动力学的影响还不是很清楚。为了研究表面电荷优化的动力学后果，我们对一个包含五个氨基酸取代的 Fyn SH3 结构域变体的折叠动力学进行了特征描述，该变体是通过计算设计来优化表面电荷-电荷相互作用的。我们的结果表明，这种优化的 Fyn SH3 结构域主要通过折叠速率提高八倍来稳定。对组成的单个氨基酸取代的分析表明，优化电荷-电荷相互作用对折叠速率的影响是累加的。这与折叠态稳定性中观察到的趋势相反，表明静电相互作用在过渡态中比在折叠态中不那么具体。使用粗粒度链模型对过渡态进行模拟表明，天然静电接触较弱，从而使过渡态有利于非特异性，甚至非天然的静电相互作用。由于从小蛋白的无规卷曲状态到折叠过渡状态的折叠伴随着电荷密度的增加，非特异性静电相互作用，即通用电荷密度效应，可能对蛋白质折叠动力学有显著贡献。因此，对表面带电位置氨基酸取代的影响的解释可能会变得复杂，仅考虑天然状态下的相互作用可能无法提供充分的描述。
