Submillisecond protein folding kinetics studied by ultrarapid mixing
Submillisecond protein folding kinetics studied by ultrarapid mixing
复制标题
DOI：
10.1073/pnas.94.5.1779
复制
发表时间：
1997-03-04
影响因子：
11.1
通讯作者：
Hofrichter, J
中科院分区：
文献类型：
--
作者：
Chan, CK;Hu, Y;Hofrichter, J
发展了一种研究化学变性蛋白质亚毫秒折叠的超快速混合连续流动方法，通过小间隙泵送溶液产生的湍流使变性剂在几十微秒内稀释。我们用这种方法研究了细胞色素c折叠动力学，在80亩S到3毫秒的时间范围内，为了消除通过捕捉错误折叠结构而使折叠动力学复杂化和减慢的血红素配体交换化学，用咪唑络合物进行了测量。荧光猝灭是由于激发能量从色氨酸转移到血红素，用来监测这些基团之间的距离，荧光减弱是双相的。在最低变性剂浓度(0.2M盐酸胍)下，有一个未分解的过程，即50亩S，然后是一个较慢的指数过程，其tau=600亩S。这些动力学被解释为在较低的变性剂浓度下无障碍的部分坍塌到新的平衡展开状态，然后是Stower跨越分隔展开和折叠状态的自由能垒，结果提出了关于蛋白质折叠中坍塌和跨越障碍的动力学的几个基本问题。
An ultrarapid-mixing continuous-flow method has been developed to study submillisecond folding of chemically denatured proteins, Turbulent flow created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds, We have used this method to study cytochrome c folding kinetics in the previously inaccessible time range 80 mu s to 3 ms, To eliminate the heme-ligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups, The fluorescence decrease is biphasic. There is an unresolved process with tau < 50 mu s, followed by a slower, exponential process with tau = 600 mu s at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by stower crossing of a free energy barrier separating the unfolded and folded states, The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.