Stern-Volmer modeling of steady-state Forster energy transfer between dilute, freely diffusing membrane-bound fluorophores
Stern-Volmer modeling of steady-state Forster energy transfer between dilute, freely diffusing membrane-bound fluorophores
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DOI：
10.1063/1.2800564
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发表时间：
2007-12-07
影响因子：
4.4
通讯作者：
Kamburov, Dobromir
中科院分区：
文献类型：
--
作者：
Buboltz, Jeffrey T.;Bwalya, Charles;Kamburov, Dobromir
使用两种不同的指标来评估稳态下荧光团之间的福斯特共振能量转移 (FRET)：(i) 供体荧光 E 的受体淬灭（也称为转移效率）和 (ii) 供体激发的受体荧光 F(A)(Dex)。虽然 E 的使用仍然更广泛，但 F(A)(Dex) 出于实际原因在研究生物膜的实验学家中越来越受欢迎。在这里，对于膜结合荧光团的特殊情况，我们提出了大量的实验证据，证明在稀释探针条件下对任一 FRET 指标进行建模时使用简单的 Stern-Volmer 表达式是合理的。我们还发现了 E 和 Wolber 的 Stern-Volmer 表达式与二维 (2D) 稳态福斯特淬火的 Hudson 级数近似之间的稀态对应关系。这种新颖的对应关系使我们能够根据（i）有效福斯特距离和（ii）两个最大受体浓度限制来解释每个二维猝灭常数，每个限制都定义了自己有用的实验方案。综上所述，我们的结果提出了一个三步策略，旨在设计更有效的稳态 FRET 实验来研究生物膜。 (c) 2007 年美国物理研究所。
Two different metrics are used to assess Forster resonance energy transfer (FRET) between fluorophores in the steady state: (i) acceptor-quenching of donor fluorescence E (also known as transfer efficiency) and (ii) donor-excited acceptor fluorescence F(A)(Dex). While E is still more widely used, F(A)(Dex) has been gaining in popularity for practical reasons among experimentalists who study biomembranes. Here, for the special case of membrane-bound fluorophores, we present a substantial body of experimental evidence that justifies the use of simple Stern-Volmer expressions when modeling either FRET metric under dilute-probe conditions. We have also discovered a dilute-regime correspondence between our Stern-Volmer expression for E and Wolber and Hudson's series approximation for steady-state Forster quenching in two dimensions (2D). This novel correspondence allows us to interpret each of our 2D quenching constants in terms of both (i) an effective Forster distance and (ii) two maximum acceptor-concentration limits, each of which defines its own useful experimental regime. Taken together, our results suggest a three-step strategy toward designing more effective steady-state FRET experiments for the study of biomembranes. (c) 2007 American Institute of Physics.