Bhattacharyya R P, Sosnick T R
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637, USA.
Biochemistry. 1999 Feb 23;38(8):2601-9. doi: 10.1021/bi982209j.
We measured whether solvent viscosity, and hence chain diffusion, plays a role in the rate-limiting step of the folding reactions of GCN4-p2', a simple alpha-helical coiled coil derived from the leucine zipper region of bZIP transcriptional activator GCN4. To deconvolute the dual effects of viscosogenic solvents on both viscosity, eta, and stability, earlier attempts assumed that the cosolvent and denaturant interact to the same degree in the transition state. Applying this analysis to GCN4-p2' yielded a nearly 1/eta dependence between folding rates and viscosity for both the dimeric and the cross-linked, monomeric versions of the coiled coil, but it revealed no such coherent relationship for cytochrome c. We also developed a method to determine the relative viscosity dependence of the dimeric and monomeric forms of the coiled coil independent of the assumption concerning the transition state's relative interaction with cosolvents and denaturants. Application of this method indicated that the effect of viscosity on both the folding and the unfolding rates was the same for the dimeric and monomeric versions, further supporting the view that the folding of the dimeric version is folding-limited rather than encounter-limited. The finding that GCN4-p2' folding appears to exhibit a 1/eta viscosity dependence implies that the rate-limiting step in folding is opposed predominantly by solvent-derived rather than internal frictional forces. These results are interpreted in relation to various models for protein folding.
我们测定了溶剂粘度，进而测定了链扩散，是否在GCN4-p2'折叠反应的限速步骤中起作用。GCN4-p2'是一种简单的α-螺旋卷曲螺旋，源自bZIP转录激活因子GCN4的亮氨酸拉链区域。为了剖析致粘溶剂对粘度（η）和稳定性的双重影响，早期的研究假设助溶剂和变性剂在过渡态中的相互作用程度相同。将这种分析应用于GCN4-p2'，对于卷曲螺旋的二聚体形式和交联单体形式，均得出折叠速率与粘度之间存在近似1/η的依赖关系，但对于细胞色素c却未揭示出这种连贯的关系。我们还开发了一种方法，用于确定卷曲螺旋二聚体和单体形式的相对粘度依赖性，而无需假设过渡态与助溶剂和变性剂的相对相互作用。应用该方法表明，粘度对二聚体和单体形式的折叠速率与解折叠速率的影响是相同的，这进一步支持了二聚体形式的折叠是折叠受限而非相遇受限的观点。GCN4-p2'折叠似乎呈现出1/η粘度依赖性这一发现意味着，折叠过程中的限速步骤主要受到溶剂衍生的力而非内摩擦力的阻碍。这些结果结合各种蛋白质折叠模型进行了解释。
