A method for photoinitating protein folding in a nondenaturing environment
A method for photoinitating protein folding in a nondenaturing environment
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DOI：
10.1021/ja002949r
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发表时间：
2000-11-22
影响因子：
15
通讯作者：
Chan, SI
中科院分区：
文献类型：
--
作者：
Hansen, KC;Rock, RS;Chan, SI
蛋白质折叠的早期动力学过程是蛋白质折叠途径的重要组成部分，但人们对这一过程的认识还很有限。从这些早期事件的研究中获得的信息可以让我们区分已经提出的描述蛋白质真实的折叠的各种模型。与具有明确定义的初始和最终状态的“典型”化学动力学不同，变性蛋白质的初始状态相对不明确。这种不确定性在解释蛋白质折叠早期事件的实验数据时引入了模糊性。为了建立一个统一的蛋白质折叠理论，有必要从一个定义明确的初始状态开始观察蛋白质的折叠过程，尽可能快地触发折叠，并在真实的时间内跟踪蛋白质在其高度复杂的自由能景观上的构象空间。传统的停流方法已经被用来跟踪蛋白质折叠反应的动力学过程。然而，这样的实验在时间分辨率方面受到限制，并且因为使用外部变性剂来实现非天然状态。目前尚不清楚变性剂如何与蛋白质相互作用以及它们如何影响动力学途径。此外，在这些停流实验中，在混合的死区时间期间发生大量的再折叠。为了规避时域限制，几个小组最近开发了基于温度、1 pH 2或压力跳跃、3血红素配体的闪光光解、4金属蛋白的光还原、5和工程二硫化物的光解的策略6（最近的综述见参考文献7、8）。虽然上述技术提供了深入了解蛋白质折叠，很少有研究已经在变性剂的情况下进行。更详细地研究折叠的早期事件的一般方法需要不可逆性、对非金属蛋白的适用性、快速触发和消除外部变性剂。我们的策略是基于皮秒闪光光解的有机交联剂的蛋白质折叠的开始。这样的光触发剂可以放置在感兴趣的蛋白质的环状形式内。如果选择正确，所产生的环构象将阻止蛋白质的正确折叠（图1）。因此，代替操纵外部条件，例如温度、pH，
The early kinetic events of protein folding are an important part of the folding pathway, yet our understanding towards the process is limited. Information from the study of these early events can allow us to distinguish between the various models that have been proposed to describe the folding of a protein in real time. Unlike “typical” chemical kinetics with well-defined initial and final states, the initial state of a denatured protein is relatively ill-defined. This uncertainty introduces ambiguity in the interpretation of the experimental data on the early events in protein folding. Toward developing a unified theory of protein folding, it is necessary to begin the observation of the refolding process from a well-defined initial state, trigger folding as rapidly as possible, and to follow the protein in real time as it samples its conformational space over its highly complex free-energy landscape.Traditional stopped-flow methods have been employed to follow the kinetic course of protein-folding reactions. However, such experiments are limited in time resolution and in that external denaturants are used to achieve a non-native state. It is not known exactly how denaturants interact with a protein and how they affect the kinetic pathways. In addition, substantial refolding occurs during the dead time of mixing in these stopped-flow experiments. To circumvent the time domain limitation, several groups have recently developed strategies based on temperature, 1 pH2 or pressure jumps, 3 flash photolysis of heme ligands, 4 photoreduction of metalloproteins, 5 and the photolysis of engineered disulfides6 (for a recent review see refs 7, 8). Although the above techniques have provided insight into protein folding, few studies have been done in the absence of denaturant. A general method to study the early events of folding in greater detail requires irreversibility, applicability to non-metalloproteins, fast triggering, and the elimination of external denaturants. Our strategy for the initiation of protein folding is based on the picosecond flash photolysis of an organic cross-linker. Such a phototrigger may be placed within a cyclic form of a protein of interest. If chosen correctly, the resulting loop conformation will prohibit proper folding of the protein (Figure 1). Thus, instead of manipulating the external conditions, such as temperature, pH,