Nonlinear free energy relationships in arc repressor unfolding imply the existence of unstable, native-like folding intermediates
Nonlinear free energy relationships in arc repressor unfolding imply the existence of unstable, native-like folding intermediates
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DOI：
10.1021/bi953056s
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发表时间：
1996-04-16
期刊：
影响因子：
2.9
通讯作者：
Sauer, RT
中科院分区：
文献类型：
--
作者：
Jonsson, T;Waldburger, CD;Sauer, RT
在强变性条件下，野生型Are二聚体的解离/展开的速率常数的对数随变性剂浓度以非线性方式变化。为了评估在有利于天然结构的条件下的解折叠/解离行为，我们混合了用荧光受体或供体基团标记的Are变体，并使用能量转移来监测异二聚体随时间的增加。在本实验的条件下，异二聚体浓度接近其平衡值的速率由蛋白质的解离和解折叠的速率决定。使用这种方法和传统的变性剂跳跃实验，展开/解离的速率常数确定在广泛的稳定和不稳定的条件。在每种情况下检查，日志（k（u））与变性剂的曲线图显示出显着的曲率在强烈的变性条件下，即使其他动力学实验表明，展开/解离反应仍然主要是两个状态。这种曲率可以最容易地解释由一系列不稳定的中间体在展开途径中，与变性剂诱导的变化的动力学步骤是限速的，或者，曲率可能导致从哈蒙德行为，其中的过渡态的结构变得更天然的，如天然的Are的稳定性随着变性剂的增加而降低。
Under strongly denaturing conditions, the logarithm of the rate constant for dissociation/unfolding of the wild-type Are dimer varies in a nonlinear fashion with denaturant concentration. To assess the unfolding/dissociation behavior under conditions favoring the native structure, we mixed Are variants labeled with fluorescence acceptor or donor groups and used energy transfer to monitor the increase in heterodimer with time. Under the conditions of this experiment, the rate at which the heterodimer concentration approaches its equilibrium value is determined by the rate of dissociation and unfolding of the protein. Using this method and traditional denaturant-jump experiments, rate constants for unfolding/dissociation were determined over a wide range of stabilizing and destabilizing conditions. In each case examined, plots of log(k(u)) versus denaturant showed significant curvature under strongly denaturing conditions, even though other kinetic experiments indicate that the unfolding/dissociation reactions remain largely two-state. This curvature can be explained most readily by a series of unstable intermediates in the unfolding pathway, with denaturant-induced changes in the kinetic step that is rate-limiting, Alternatively, curvature might result from Hammond behavior in which the structure of the transition state becomes more native-like as the stability of native Are decreases with increasing denaturant.