Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins
Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins
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DOI：
10.1073/pnas.0508584102
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发表时间：
2005-11-29
影响因子：
11.1
通讯作者：
Weiss, S
中科院分区：
文献类型：
--
作者：
Laurence, TA;Kong, XX;Weiss, S
我们利用单分子FRET和交变激光激发研究蛋白质和核酸的结构和动力学。根据在100 μ s至1 ms内检测到的单分子信号，将自由扩散的分子分类为亚种群。利用时间相关的单光子计数，研究了这些亚种群中由大于100 μ s的波动引起的距离分布。dsDNA的测量距离分布可以通过考虑荧光团连接物和荧光团旋转扩散来解释，除了我们发现内部标记的dsDNA比位于末端位置的一个荧光团的DNA波动更小。我们发现，短单链poly（dT）的持续长度的静电部分大致随离子强度(l)的-1/2次方（l(-1/2)）而变化，并且对每个碱基轮廓长度的平均贡献为0.40-0.45 nm。我们研究了未折叠的胰凝乳蛋白酶抑制剂2（02）和未折叠的酰基辅酶a结合蛋白（ACBP），即使在折叠亚群和未折叠亚群共存的条件下（使用交替激光激发排除折叠蛋白的贡献）。在较低的变性剂浓度下，未折叠的C12和ACBP比只存在未折叠蛋白的较高变性剂浓度下更紧凑，波动更大。实验测量的波动比高斯链模型或虫状链模型预测的波动要大。我们提出较大的波动可能表明未展开状态下的瞬态残余结构。
We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on single-molecule signals detected within 100 mu s to 1 ms. Distance distributions caused by fluctuations faster than 100 mu s are studied within these subpopulations by using time-correlated single-photon counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (l) to the -1/2 power (l(-1/2)), and that the average contribution to the contour length per base is 0.40-0.45 nm. We study unfolded chymotrypsin inhibitor 2 (02) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded C12 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state.