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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Effect of flexibility and cis residues in single-molecule FRET studies of polyproline
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Effect of flexibility and cis residues in single-molecule FRET studies of polyproline

机译：柔性和顺式残基在聚脯氨酸单分子FRET研究中的作用

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摘要

Polyproline has recently been used as a spacer between donor and acceptor chromophores to help establish the accuracy of distances determined from single-molecule Forster resonance energy transfer (FRET) measurements. This work showed that the FRET efficiency in water is higher than expected for a rigid spacer and was attributed to the flexibility of the polypeptide. Here, we investigate this issue further, using a combination of single-molecule fluorescence intensity and lifetime measurements, NMR, theory, and molecular dynamics simulations of polyproline-20 that include the dyes and their linkers to the polypeptide. NMR shows that in water ≈ 30% of the molecules contain internal cis prolines, whereas none are detectable in trifluoroethanol. Simulations suggest that the all-trans form of polyproline is relatively stiff, with persistence lengths of 9-13 nm using different established force fields, and that the kinks arising from internal cis prolines are primarily responsible for the higher mean FRET efficiency in water. We show that the observed efficiency histograms and distributions of donor fluorescence lifetimes are explained by the presence of multiple species with efficiencies consistent with the simulations and populations determined by NMR. In calculating FRET efficiencies from the simulation, we find that the fluctuations of the chromophores, attached to long flexible linkers, also play an important role. A similar simulation approach suggests that the flexibility of the chromophore linkers is largely responsible for the previously unexplained high value of R_0 required to fit the data in the classic study of Stryer and Haugland.
机译：聚脯氨酸最近已用作供体和受体生色团之间的间隔基，以帮助确定由单分子福斯特共振能量转移（FRET）测量确定的距离的准确性。这项工作表明水中的FRET效率比刚性间隔子要高，这归因于多肽的柔韧性。在这里，我们结合使用单分子荧光强度和寿命测量，NMR，理论和聚脯氨酸20的分子动力学模拟（包括染料及其与多肽的连接）相结合，进一步研究该问题。 NMR显示，在水中约有30％的分子含有内部顺式脯氨酸，而在三氟乙醇中则没有检测到。模拟表明，聚脯氨酸的全反式相对较硬，使用不同的既定力场的持久长度为9-13 nm，内部顺式脯氨酸产生的扭结是水中平均FRET效率较高的主要原因。我们表明，观察到的效率直方图和供体荧光寿命的分布是由多种物质的存在所解释的，这些物质的效率与通过NMR确定的模拟和种群一致。在通过仿真计算FRET效率时，我们发现附着在长柔性接头上的生色团的波动也起着重要作用。一种类似的模拟方法表明，发色团接头的柔性在很大程度上决定了以往无法解释的R_0的高值，该值需要对Stryer和Haugland的经典研究进行拟合。

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