pH-induced folding/unfolding of staphylococcal nuclease: determination of kinetic parameters by the sequential-jump method.
pH-induced folding/unfolding of staphylococcal nuclease: determination of kinetic parameters by the sequential-jump method.
复制标题
pH 诱导的葡萄球菌核酸酶折叠/解折叠：通过顺序跳跃法测定动力学参数。
DOI：
10.1021/bi00120a027
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发表时间：
1992
期刊：
影响因子：
2.9
通讯作者：
Tsong,TY
中科院分区：
文献类型：
--
作者：
Chen,HM;Markin,VS;Tsong,TY
明尼苏达大学生物科学学院生物化学系，圣保罗，明尼苏达州55108，1991年6月18日接收; 1991年10月25日接收修订版摘要：在已有的停流pH跃变实验的基础上，我们提出酸和碱诱导的葡萄球菌核酸酶折叠/去折叠转变，在2 ms ~ 300 s的时间范围内，遵循路径N 0 → D1 → D2 → D3，其中D1、D2和D3是未折叠状态的三个子状态，N 0是天然状态。停流“双跳”技术已被用来测试这种机制，并确定由于缺乏荧光信号而无法通过直接pH跳变获得的速率常数，即D1转化为D2和D2转化为D3的速率。在前向跳跃中，将保持在pH 7.0的蛋白质溶液与酸性或碱性溶液混合，分别达到最终pH 3.0或12.2。将混合溶液保持不同的时间段，称为延迟时间，ID。启动第二次混合（后跳）以使蛋白质溶液回到pH 7.0。分析在回跳中恢复的Trp-140荧光信号的时间过程作为fD的函数。展开的动力学被发现是三相的双跳法，相反的直接pH值跳跃观察到的双相动力学。复杂的动力学展开预期与建议的动力学计划。通过直接跳跃和双跳跃实验获得的数据的分析，得到了完整的速率常数和活化能的展开到pH 3.0和pH 12.2和foldingfrom酸和碱pH 7.0。在pH 7.0时，N 0、Db、D2和D3状态的蛋白质分数分别为1.0、0、0和0;在pH 3.0时，分别为0、0.61、0.28和0.11;在pH 12.2时，分别为0、0.43、0.30和0.27。三个D的任何两个相邻物之间的转变的Δ G小于0.55 kcal mol-1。因此，在链折叠的早期阶段，任何强制性途径都必须由动力学障碍决定，而不是由中间状态的稳定性决定。葡萄球菌核酸酶（Staphylococcal nuclease，SNase）是研究蛋白质折叠的经典蛋白质。首先，它由一条肽链组成，是一种分子量相对较小的单域蛋白质，分子量为16800（149个残基）。因此，它的稳定性和折叠途径应该比结构更复杂的蛋白质更适合生物物理学研究。第二，没有二硫化物
Department of Biochemistry, University of Minnesota College of Biological Sciences, St. Paul, Minnesota 55108 Received June 18, 1991; Revised Manuscript Received October 25, 1991 abstract: On the basis of previous stopped-flow pH-jump experiments, we have proposed that the acidand alkaline-induced folding/unfolding transition of staphylococcal nuclease, in the time range 2 ms to 300 s, follows the pathway N0^ Dt^ D2^ D3, in which D,, D2, and D3 are three substates of the unfolded state and N0 is the native state. The stopped-flow “double-jump” technique has been employed to test this mechanism and to determine the rate constants which would not be accessible by the direct pH jump because of the lack of fluorescence signal, ie, the rates for the conversion of Dj to D2 and of D2 to D3. In the forward jump, a protein solution kept at pH 7.0 was mixed with an acidic or alkaline solution to the final pH of 3.0 or 12.2, respectively. The mixed solution was kept for varying periods of time, called the delay time, íD. A second mixing (the back jump) was launched to bring the protein solution back to pH 7.0. The time course of the Trp-140 fluorescence signals recovered in the back jump was analyzed as a function of/D. Kinetics of the unfolding were found to be triphasic by the double-jump method, contrary to the monophasic kinetics observed by the direct pH jump. Complex kinetics of unfolding are expected with the proposed kinetic scheme. Analysis of data obtained by both the direct-jump and the double-jump experiments yielded complete sets of rate constants and activation energies for the unfolding to pH 3.0 and to pH 12.2 and for the foldingfrom acid and alkali to pH 7.0. Fractions of protein in N0, Db D2, and D3 states were determined to be 1.0, 0, 0, and 0, respectively, at pH 7.0; 0, 0.61, 0.28, and 0.11, respectively, at pH 3.0; and 0, 0.43, 0.30, and 0.27, respectively, at pH 12.2. AG of the transition between any two neighbors of the three D’s was less than 0.55 kcal mol" 1. Thus, any obligatory pathway in an early stage of the chain folding must be determined by the kinetic barriers rather than by the stability of the intermediate states.Staphylococcal nuclease (SNase) is a classical protein for the study of protein folding because of several convenient features. First, it consists of one peptide chain and is a single-domain protein of relatively small molecular weight, 16800 (149 residues). Thus, its stability and pathway for folding should bemore amenable to biophysical study than proteins of more complex structure. Second, there are no disulfide