Distinguishing between Protein Dynamics and Dye Photophysics in Single-Molecule FRET Experiments
Distinguishing between Protein Dynamics and Dye Photophysics in Single-Molecule FRET Experiments
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DOI：
10.1016/j.bpj.2009.12.4322
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发表时间：
2010-02-17
影响因子：
3.4
通讯作者：
Eaton, William A.
中科院分区：
文献类型：
--
作者：
Chung, Hoi Sung;Louis, John M.;Eaton, William A.
单分子实验中的福斯特共振能量转移（FRET）效率分布包含结构和动态信息，从这些分布中提取这些信息需要仔细分析染料光物理学的贡献。为了研究FRET以外的机制如何影响通过计数供体和受体光子获得的分布，我们测量了小α / β蛋白（即蛋白质GB1）的单分子荧光轨迹，经历了两态，折叠/展开转变。将Alexa 488供体和Alexa 594受体染料分别附着在位置10和57的半胱氨酸上，得到两个在纯化过程中无法分离的异构体：供体(10)/受体（57）和供体(57)/受体（10）。通过将n端连接序列上的组氨酸标签与嵌入在聚乙烯乙二醇涂层玻璃表面的铜离子结合，将蛋白质固定。从轨迹组装的FRET效率分布是复杂的，单个峰的宽度远远超过由散粒噪声引起的宽度。这种复杂性大部分可以用两种干扰的光物理效应来解释——供体染料的光致红移和两种异构体的受体染料的量子产率的差异，这是由铜离子猝灭速率的差异造成的。稳态偏振的测量、光子轨迹的施主-受主互相关函数的计算以及单分子动力学和系综动力学的比较都表明，构象分布和动力学对复杂性没有贡献。
Forster resonance energy transfer (FRET) efficiency distributions in single-molecule experiments contain both structural and dynamical information Extraction of this information from these distributions requires a careful analysis of contributions from dye photophysics. To investigate how mechanisms other than FRET affect the distributions obtained by counting donor and acceptor photons, we have measured single-molecule fluorescence trajectories of a small alpha/beta protein, i.e, protein GB1, undergoing two-state, folding/unfolding transitions. Alexa 488 donor and Alexa 594 acceptor dyes were attached to cysteines at positions 10 and 57 to yield two isomers-donor(10)/acceptor(57) and donor(57)/acceptor(10)-which could not be separated in the purification The protein was immobilized via binding of a histidine tag added to a linker sequence at the N-terminus to cupric ions embedded in a polyethylene-glycol-coated glass surface. The distribution of FRET efficiencies assembled from the trajectories is complex with widths for the individual peaks in large excess of that caused by shot noise Most of this complexity can be explained by two interfering photophysical effects-a photoinduced red shift of the donor dye and differences in the quantum yield of the acceptor dye for the two isomers resulting from differences in quenching rate by the cupric ion. Measurements of steady-state polarization, calculation of the donor-acceptor cross-correlation function from photon trajectories, and comparison of the single molecule and ensemble kinetics all indicate that conformational distributions and dynamics do not contribute to the complexity.