Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).
Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).
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DOI：
10.1039/c0cp01911a
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发表时间：
2011-02-07
期刊：
影响因子：
--
通讯作者：
Schuler B
中科院分区：
文献类型：
--
作者：
Hoffmann A;Nettels D;Clark J;Borgia A;Radford SE;Clarke J;Schuler B
单分子Förster共振能量转移(FRET)实验是研究生物大分子构象分布和动力学的一种通用方法。在一种常见类型的实验中，当单个分子在溶液中自由扩散时，当它们穿过共焦显微镜的观察体积时，会检测到它们的荧光爆发。对荧光爆发的关联分析表明，在典型的实验条件下，在长达几十毫秒的时间尺度上，分子返回共焦体积的概率大于检测到新分子的概率。在这里，我们提出了RASP(单粒子递归分析)，这是一种基于这种递归行为的方法，允许我们显著扩展可以从单分子FRET实验中提取的信息。通过构造递归FRET效率直方图，基本上可以以一种无模型的方式识别样本中的亚总体的数量和峰形。这些是通过首先从较小的传输效率范围(初始突发)中选择光子突发，然后仅根据在初始突发之后的短时间(重复间隔)内检测到的突发构建FRET效率直方图来获得的。重复间隔的系统变化允许从平衡测量的~50μS到~100ms的时间尺度上确定亚种群之间的相互转化动力学。我们证明了该方法在几种具有不同程度构象异质性和折叠动力学的多肽和蛋白质的测量上的适用性。这里提出的概念可以扩展到从单分子荧光实验中获得的其他可观测数据。
Single molecule Förster resonance energy transfer (FRET) experiments are a versatile method for investigating the conformational distributions and dynamics of biological macromolecules. In a common type of experiment, the fluorescence bursts from individual molecules freely diffusing in solution are detected as they pass through the observation volume of a confocal microscope. Correlation analysis of the fluorescence bursts shows that under typical experimental conditions, for time scales up to several tens of milliseconds, the probability for a molecule to return to the confocal volume is greater than the probability of a new molecule being detected. Here we present RASP (recurrence analysis of single particles), a method that is based on this recurrence behavior and allows us to significantly extend the information that can be extracted from single molecule FRET experiments. The number and peak shapes of subpopulations within the sample can be identified essentially in a model-free way by constructing recurrence FRET efficiency histograms. These are obtained by first selecting photon bursts from a small transfer efficiency range (initial bursts), and then building the FRET efficiency histogram only from bursts detected within a short time (the recurrence interval) after the initial bursts. Systematic variation of the recurrence interval allows the kinetics of interconversion between subpopulations to be determined on time scales from ~50 μs up to ~100 ms from equilibrium measurements. We demonstrate the applicability of the method on measurements of several peptides and proteins with different degrees of conformational heterogeneity and folding dynamics. The concepts presented here can be extended to other observables available from single molecule fluorescence experiments.
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影响因子：
3.4
作者：
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作者：
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DOI：
10.1073/pnas.0901178106
发表时间：
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