Folding/unfolding kinetics of mutant forms of iso-1-cytochrome c with replacement of proline-71.
Folding/unfolding kinetics of mutant forms of iso-1-cytochrome c with replacement of proline-71.
复制标题
取代脯氨酸 71 的 iso-1-细胞色素 c 突变体形式的折叠/解折叠动力学。
DOI：
10.1021/bi00370a033
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Nall,BT
中科院分区：
文献类型：
--
作者：
Ramdas,L;Nall,BT
德克萨斯大学休斯敦医学院生物化学和分子生物学系，1986年2月24日收到；修订手稿收到1986年7月18日摘要：Proline-71，一个进化上保守的残基，分隔两个短螺旋区域，被来自酿酒酵母的Valine，苏氨酸，或异亮氨酸所取代，至少部分功能形式的iso-1-细胞色素c[Ernst，JF，Hampsey，D.M.，Stewart，J.W.，Rackovsky，S.，Goldstein，D.，&Sherman，F.(1985年)J.Biol]。化学。260,13225-13236]，为了确定蛋白质折叠过程中第71位的扰动对步骤的影响，比较了正常蛋白质和三种突变形式的折叠/去折叠反应的动力学性质。在pH 6.0，20℃时，利用停流混合进行盐酸胍浓度跳跃，在平衡过渡区以下、内和上方监测荧光检测的折叠/去折叠动力学。四种蛋白质中的每一种都检测到三个动力学相。对于野生型和突变蛋白，这些阶段中最快的阶段(R3)的速率不同。其余的动力学相(t‘和t2)在整个折叠/展开条件下对所有四种蛋白质都有相似的速率。盐酸胍对动力学相相对幅度的依赖是复杂的，并且对第71位取代基的性质很敏感：这四种蛋白质中的每一种都显示出与两个最快速度过程相关的折叠/去折叠比例的差异。结果表明，是一级结构中突变的位置而不是取代基的性质决定了哪个动力学步骤(或多个步骤)的变化速度。然而，动力学振幅(a2和a3)对71位取代基的性质非常敏感。因此，不同的突变对负责快速折叠阶段的物种的稳定性确实有不同的影响。与脯氨酸异构化假说的预期相反[Brandts，J.F.，Halvorson，H.R.，&Brennan，M.(1975)BioChemical 14,4953-4963]，Pro-71的取代对荧光检测到的缓慢复性几乎没有影响。用Val-71取代Pro-71既不影响缓慢折叠的幅度(a，)，也不影响时间常数(Tj)。Thr-71和Ile-71的替代都不能改变缓慢复性的速度，尽管观察到相对幅度的降低。
Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, Texas 77225 Received February 24, 1986; Revised Manuscript Received July 18, 1986 abstract: Proline-71, an evolutionally conserved residue that separates two short-helical regons, is replaced by valine, threonine, or isoleucine in at least partially functional forms of iso-1-cytochrome c from Saccharomyces cerevisiae [Ernst, JF, Hampsey, D. M., Stewart, J. W., Rackovsky, S., Goldstein, D., & Sherman, F.(1985) J. Biol. Chem. 260, 13225-13236], To assign the effects of perturbations at position 71 to steps in the process of protein folding, the kinetic properties of the folding/unfolding reactions of normal protein and the three mutant forms are compared. At pH 6.0, 20 C, fluorescence-detected folding/unfolding kinetics are monitored below, within, and above the equilibrium transition zone by using stopped-flow mixing to perform guanidine hydrochloride concentration jumps. Three kinetic phases are detected for each of the four proteins. The fastest of these phases (r3) differs inrate for the wild typeand mutant proteins. The remaining kinetic phases (t¡ and t2) have similar rates for all four proteins over the entire range of folding/unfolding conditions. The guanidine hydrochloride dependence of the relative amplitudes of the kinetic phases is complex and is sensitive to the nature of the substituent at position 71: each of the four proteins shows differences in the fraction of folding/unfolding associated with the two fastest rate processes. The results suggest that it is the location of the mutation in the primary structure rather thanthe nature of the substituentthat determines which kinetic step (or steps) is changed in rate. However, the kinetic amplitudes (a2 and a3) are very sensitive to the nature of the substituent at position 71. Thus different mutations do have different effects on the stability of the species responsible for fast folding phases. Contrary to the expectations of the proline isomerization hypothesis [Brandts, J. F., Halvorson, H. R., & Brennan, M.(1975) Biochemistry 14, 4953-4963], replacements of Pro-71 have little effect on fluorescence-detected slow refolding. Replacement of Pro-71 by Val-71 does not affect either the amplitude (a,) or time constant (tj) for slow refolding. Neither the Thr-71 nor the Ile-71 replacement alters the rate of slow refolding, although decreases in relative amplitude are observed.