Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2
Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2
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DOI：
10.1073/pnas.090104997
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发表时间：
2000-05-09
影响因子：
11.1
通讯作者：
Weiss, S
中科院分区：
文献类型：
--
作者：
Deniz, AA;Laurence, TA;Weiss, S
我们报告的小，单域蛋白质，胰凝乳蛋白酶抑制剂2（CI2）的单分子折叠研究。CI2是蛋白质折叠研究的一个很好的模型系统，已经在实验（系综水平）和理论上进行了广泛的研究。构象辅助连接方法用于合成蛋白质，并用供体和受体染料对其进行位点特异性标记。折叠和变性的亚群观察自由扩散的单蛋白分子的荧光共振能量转移（FRET）测量。直接监测这些亚群的性质作为氯化胍浓度的函数。它表明，新的信息，不同方面的蛋白质折叠反应，可以从这样的亚群属性提取。转移效率的平均值的变化进行了讨论，FRET效率分布被翻译成电位，和变性曲线直接绘制的FRET峰的面积。突变引起的稳定性变化也通过比较假野生型C12与去稳定化突变体（K17G）来测量。目前的局限性和未来的可能性和前景单对FRET蛋白质折叠的调查进行了讨论。
We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.