Probing heme protein conformational equilibration rates with kinetic selection.
Probing heme protein conformational equilibration rates with kinetic selection.
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通过动力学选择探测血红素蛋白构象平衡率。
DOI：
10.1021/bi952474u
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
Sligar,SG
中科院分区：
文献类型：
--
作者：
Tian,WD;Sage,JT;Champion,PM;Chien,E;Sligar,SG
利用双脉冲闪光光解实验确定了蛋白质构象平均的时间尺度。根据溶剂组成和温度的不同，MbCO的“开放”和“关闭”亚族之间的相互转化时间为10-6−10-4s。在273K的水溶液中，其相互转化速率为1.4×106s。由于相互转化速率相当于或慢于双态重新结合速率，我们将动力学的双态相描述为来自开态和闭态的贡献的叠加。尽管在室温附近，开态和闭态的双态动力学本质上都是非指数的，但我们发现这两个亚群中的亚态相互转化比双态重新结合更快。这些观察结果不能用准连续构象分布的贡献叠加来解释(Steinbach等人，1991)，可能是由于蛋白质弛豫的长期尾巴(Tian等人，1992)。双分子再结合以统计平均速率发生，因为相互转化和松弛速率比双分子动力学快。利用这种方法和光谱确定的开态和闭态布居，定量地分析了双键和双分子动力学随pH的变化。该分析解释了观察到的动力学，并成功地预测了在双脉冲实验中观察到的动力学响应。在273K的水溶液中，开态的双峰幅度和速率分别为=32%和1.3×107s-1，闭态的双峰幅度和速率分别为9.3%和1.4×106s-1。在264K的75%甘油溶液中，双酯结合的主要成分为：开放状态=89%和=3.1×106s-1，闭合状态=26%和=3.1×106s-1。水溶液中闭合状态的相互转化率与闭合状态的成双率相当，这一事实与开放状态为配体逃逸(或进入)血红素口袋提供了一条重要的途径(Tian等人，1993)。75%甘油溶液粘度的增加会延迟闭合的→开放相互转化，直到双酯化阶段结束，这迫使配体寻找替代的途径来获得溶液。这一观察结果，以及在75%甘油溶液中开放和关闭状态的配对率几乎相同，表明溶剂组成从根本上改变了蛋白质−配体的动力学。
Double-pulse flash photolysis experiments on solutions of carbonmonoxymyoglobin (MbCO) are used to determine the time scale for protein conformational averaging. The interconversion times for transitions between the “open” and “closed” subpopulations of MbCO are found to be 10-6−10-4s, depending on solvent composition and temperature. In aqueous solution at 273 K, the interconversion rate is found to be 1.4 × 106s. Since the interconversion rate is comparable to or slower than the geminate rebinding rate, we describe the geminate phase of the kinetics as a superposition of contributions from the open and closed states. Although geminate kinetics remain intrinsically nonexponential for both open and closed states near room temperature, we find that substates within these two subpopulations interconvert more rapidly than the geminate rebinding. These observations cannot be explained by a superposition of contributions from a quasicontinuous conformational distribution (Steinbach et al., 1991) and are probably due to the long-time tail of the relaxation of the protein (Tian et al., 1992). Bimolecular rebinding takes place at a statistically averaged rate, since the interconversion and relaxation rates are faster than the bimolecular kinetics. The geminate and bimolecular kinetics are analyzed quantitatively as a function of pH using this approach and the spectroscopically determined populations of the open and closed states. The analysis accounts for the observed kinetics and also successfully predicts the kinetic response observed in the double-pulse experiments. In aqueous solution at 273 K, the geminate amplitudes and rates are found to be = 32% and = 1.3 × 107s-1for the open state and = 9.3% and = 1.4 × 106s-1for the closed state. In 75% glycerol solution at 264 K, the dominant component of the geminate rebinding is characterized by = 89% and = 3.1 × 106s-1for the open state and = 26% and = 3.1 × 106s-1for the closed state. The fact that the interconversion rate is comparable to the geminate rate of the closed state in aqueous solution is consistent with the idea that the open state provides an important pathway for ligand escape from (or entry to) the heme pocket (Tian et al., 1993). The increased viscosity of 75% glycerol solution delays the closed → open interconversion until the end of the geminate phase, which forces the ligand to find alternative pathways to the solution. This observation, in conjunction with the near equivalence of the geminate rates for the open and closed states in 75% glycerol solution, suggests that the solvent composition fundamentally alters the protein−ligand dynamics.