Photobleaching pathways in single-molecule FRET experiments
Photobleaching pathways in single-molecule FRET experiments
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DOI：
10.1021/ja068002s
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发表时间：
2007-04-18
影响因子：
15
通讯作者：
Weiss, Shimon
中科院分区：
文献类型：
--
作者：
Kong, Xiangxu;Nir, Eyal;Weiss, Shimon
为了利用单分子荧光共振能量转移(sm-fret)获得复杂生物分子机器的精确结构和动力学信息，需要大量的施主和受主光子。为了获得这样的通量，可以使用更高的激光激发强度；然而，这会导致光漂白速率的增加。抗氧剂已被广泛用于减少受主的光漂白。在这里，我们重点破译光漂白途径的最初步骤。利用最近发展起来的一系列单分子和系综光谱以及双标记的酰辅酶A结合蛋白和双链DNA作为模型系统，我们研究了这些光漂白途径，这些途径给sm-fret实验带来了根本的限制。我们发现：(I)受体光漂白膜具有FRET效率，(Ii)受体光漂白在皮秒脉冲(VS连续波)激发下得到增强，(Iii)受体光漂白膜仅以短波长(施主)激发激光的强度增强。根据这些发现，我们推断，受体光漂白的主要途径是通过吸收受体第一激发单重态的短波长光子，而供体的光漂白通常不是一个问题。最后，我们建议使用短脉冲来激发施主，以及其他可能的补救措施，以减少SMRET测量中受主的光漂白。
To acquire accurate structural and dynamical information on complex biomolecular machines using single-molecule fluorescence resonance energy transfer (sm-FRET), a large flux of donor and acceptor photons is needed. To achieve such fluxes, one may use higher laser excitation intensity; however, this induces increased rates of photobleaching. Anti-oxidant additives have been extensively used for reducing acceptor's photobleaching. Here we focus on deciphering the initial step along the photobleaching pathway. Utilizing an array of recently developed single-molecule and ensemble spectroscopies and doubly labeled Acyl-CoA binding protein and double-stranded DNA as model systems, we study these photobleaching pathways, which place fundamental limitations on sm-FRET experiments. We find that: (i) acceptor photobleaching scales with FRET efficiency, (ii) acceptor photobleaching is enhanced under picosecond-pulsed (vs continuous-wave) excitation, and (iii) acceptor photobleaching scales with the intensity of only the short wavelength (donor) excitation laser. We infer from these findings that the main pathway for acceptor's photobleaching is through absorption of a short wavelength photon from the acceptor's first excited singlet state and that donor's photobleaching is usually not a concern. We conclude by suggesting the use of short pulses for donor excitation, among other possible remedies, for reducing acceptor's photobleaching in sm-FRET measurements.