Exploring protein structure and dynamics under denaturing conditions by single-molecule FRET analysis.-猫眼课题宝

通过单分子 FRET 分析探索变性条件下的蛋白质结构和动力学。

DOI：

10.1002/mabi.200600158

发表时间：

2006

期刊：

Macromolecular bioscience

影响因子：

4.6

通讯作者：

中科院分区：

工程技术3区

文献类型：

作者：

G. Nienhaus

蛋白质是高度复杂的生物聚合物，在其正确折叠的天然状态下表现出相当程度的结构变异性。在变性剂的存在下，这种异质性大大增强，并且通过许多不同的途径在大量的折叠和未折叠构象之间发生波动。为了更好地理解蛋白质折叠，有必要探索折叠和未折叠的多肽链的结构和能量特性，以及该链通过其多维构象能量景观导航的轨迹沿着。近年来，单分子荧光光谱已被确立为这一研究领域的一个强大的工具，因为它允许一个监测的结构和动态的单个多肽链在真实的时间与原子尺度分辨率使用福斯特共振能量转移（FRET）。因此，可以直接观察到折叠转变的时间轨迹，包括可能存在于沿着这些途径的瞬态中间体。在这里，我们说明了单分子荧光的力量与我们最近的工作的结构和动力学的小酶RNase H的化学变性剂氯化胍（GdmCl）的存在下。对于FRET分析，将一对荧光染料连接到酶的特定位置。为了观察单个蛋白质分子的构象变化长达数百秒，蛋白质被固定在纳米结构的聚合物涂层玻璃表面上，该表面专门开发为与折叠和未折叠的蛋白质具有可忽略的相互作用。单分子FRET分析提供了深入了解响应于不同的变性剂浓度的未折叠的多肽链的结构变化，并且时间轨迹揭示了FRET水平的逐步转变，反映了构象动力学。在RNase H的自由能景观的障碍估计从动力学的转换。

Proteins are highly complex biopolymers, exhibiting a substantial degree of structural variability in their properly folded, native state. In the presence of denaturants, this heterogeneity is greatly enhanced, and fluctuations take place among vast numbers of folded and unfolded conformations via many different pathways. To better understand protein folding it is necessary to explore the structural and energetic properties of the folded and unfolded polypeptide chain, as well as the trajectories along which the chain navigates through its multi-dimensional conformational energy landscape. In recent years, single-molecule fluorescence spectroscopy has been established as a powerful tool in this research area, as it allows one to monitor the structure and dynamics of individual polypeptide chains in real time with atomic scale resolution using Förster resonance energy transfer (FRET). Consequently, time trajectories of folding transitions can be directly observed, including transient intermediates that may exist along these pathways. Here we illustrate the power of single-molecule fluorescence with our recent work on the structure and dynamics of the small enzyme RNase H in the presence of the chemical denaturant guanidinium chloride (GdmCl). For FRET analysis, a pair of fluorescent dyes was attached to the enzyme at specific locations. In order to observe conformational changes of individual protein molecules for up to several hundred seconds, the proteins were immobilized on nanostructured, polymer coated glass surfaces specially developed to have negligible interactions with folded and unfolded proteins. The single-molecule FRET analysis gave insight into structural changes of the unfolded polypeptide chain in response to varying the denaturant concentration, and the time traces revealed stepwise transitions in the FRET levels, reflecting conformational dynamics. Barriers in the free energy landscape of RNase H were estimated from the kinetics of the transitions.