Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions
Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions
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DOI：
10.1073/pnas.0507728102
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发表时间：
2005-10-25
影响因子：
11.1
通讯作者：
Nienhaus, GU
中科院分区：
文献类型：
--
作者：
Kuzmenkina, EV;Heyes, CD;Nienhaus, GU
蛋白质是高度复杂的系统，在其折叠状态下表现出相当程度的结构可变性。在变性剂的存在下，非均质性大大增强，大量折叠和未展开构象之间的波动通过许多不同的途径发生。在这里，我们使用单分子荧光显微镜研究了化学变性剂氯化胍（GdmCl）存在下小酶核糖核酸酶HI （RNase H）的结构和动力学，特别关注了展开态系综的表征。通过福斯特共振能量转移（FRET），将染料对特异性地附着在酶上，以测量结构变化。星形聚合物表面的酶固定化是专门为与折叠和未折叠蛋白质的可忽略的相互作用而开发的，使我们能够在几百秒内监测单个蛋白质的构象变化。从共聚焦扫描图像计算FRET效率直方图。结果显示，随着GdmCl浓度的增加，未折叠蛋白的数量增加。单分子供体和受体荧光强度时间谱的互相关分析显示，在1.7 M GdmCl下，多肽链在大约20 μ s的时间尺度上发生了重构。缓慢的构象动力学引起了特征的、逐步的FRET效率变化。折叠和未折叠酶分子之间的转变发生在100秒的时间尺度上，与体积变性实验非常一致。未展开构象之间的转换更频繁，特征时间约为2s。对这些数据进行了分析，以获得在化学变性剂存在下RNase H的自由能景观的信息。
Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Forster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of approximate to 20 mu s at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of approximate to 2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.