Goldberg J M, Baldwin R L
Department of Biochemistry, Beckman Center, Stanford University Medical Center, Stanford, California 94305-5307, USA.
Biochemistry. 1998 Feb 24;37(8):2556-63. doi: 10.1021/bi972403q.
The effects of mutations, temperature, and solvent viscosity on the bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex (RNaseS*) formed by S-peptide analogues and folded S-protein are reported. An important advantage of this system is that both kon and koff may be measured under identical strongly native conditions, and Kd for the complex may be calculated from the ratio koff/kon (preceding article). The side chains of S-peptide residues His-12 and Met-13 contribute a large fraction of the total interface with S-protein. Changing these residues, either singly or in a double mutant, destabilizes RNaseS* by up to 6 orders of magnitude, but causes no more than a 3-fold decrease in kon. Therefore, nativelike side-chain interactions between these residues and S-protein are not present in the transition state for folding. The absence of side-chain interactions in the transition state is surprising, since it has buried 55% of the total surface area that is buried upon forming RNaseS*, as estimated from the denaturant dependences of kon and koff (preceding article). The temperature dependence of the refolding rate suggests that the transition state for complex formation is stabilized by hydrophobic interactions: 66% of the change in heat capacity on forming RNaseS* occurs in the association reaction, consistent with the estimate of surface area burial from the denaturant studies. The solvent viscosity is varied to determine if the folding reaction is diffusion limited. Because kon, koff, and Kd all can be measured under the same native conditions, the viscosity effect on reaction rates can be separated from the effect of sucrose on the stability of RNaseS*. Both kon and koff are found to be inversely proportional to the solvent viscosity, indicating that the association and dissociation kinetics are diffusion controlled. The stabilizing effect of sucrose on RNaseS* appears as a reduction in koff.
报道了突变、温度和溶剂粘度对由S-肽类似物和折叠的S-蛋白形成的复合物（RNaseS*）的双分子缔合速率常数（kon）和解离速率常数（koff）的影响。该系统的一个重要优点是kon和koff都可以在相同的强天然条件下测量，并且复合物的Kd可以从koff/kon的比值计算得出（前文）。S-肽残基His-12和Met-13的侧链在与S-蛋白的总界面中占很大比例。单独或在双突变体中改变这些残基，会使RNaseS的稳定性降低多达6个数量级，但导致kon最多降低3倍。因此，这些残基与S-蛋白之间类似天然的侧链相互作用在折叠的过渡态中不存在。过渡态中不存在侧链相互作用令人惊讶，因为根据kon和koff对变性剂的依赖性估计（前文），在形成RNaseS时它掩埋了总表面积的55%。复性速率的温度依赖性表明，复合物形成的过渡态通过疏水相互作用得以稳定：形成RNaseS时热容量变化的66%发生在缔合反应中，这与变性剂研究中表面积掩埋的估计一致。改变溶剂粘度以确定折叠反应是否受扩散限制。由于kon、koff和Kd都可以在相同的天然条件下测量，因此可以将粘度对反应速率的影响与蔗糖对RNaseS稳定性的影响区分开来。发现kon和koff都与溶剂粘度成反比，表明缔合和解离动力学受扩散控制。蔗糖对RNaseS*的稳定作用表现为koff的降低。
