National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, Maryland, United States of America.
PLoS One. 2009 Nov 25;4(11):e8031. doi: 10.1371/journal.pone.0008031.
Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (k(D-->A)) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.
METHODOLOGY/PRINCIPAL FINDINGS: The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.
CONCLUSIONS/SIGNIFICANCE: We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.
Förster 共振能量转移（FRET）是一种能量通过非辐射偶极-偶极相互作用从激发供体荧光团转移到相邻发色团的机制。FRET 理论主要考虑单个供体-受体对的相互作用。不幸的是，在分子复合物中，通常不知道是否只有一个受体存在。因此，将 FRET 用作测量活细胞内蛋白质-蛋白质相互作用的工具需要了解 FRET 如何随多个受体而变化。当存在多个 FRET 受体时，假定量子能量要么从供体释放，要么全部转移到存在的一个受体。在不存在其他受体的情况下，可以测量供体与特定受体之间的能量转移速率（k(D-->A)），并且可以使用简单的动力学模型来使用这些单个 FRET 转移速率来预测总体 FRET 效率，其中所有 FRET 转移速率的总和除以所有辐射和非辐射转移速率的总和。
方法/主要发现：通过测量两种构建体中的总体 FRET 效率来测试该方法的通用性，每种构建体都包含一个单一的荧光蛋白供体（Cerulean）和两个或三个 FRET 受体（Venus）。通过系统引入点突变来消除所有其他受体，从测量的 FRET 效率中计算出这些构建体中各个供体-受体对之间的 FRET 转移速率。我们发现，在具有多个受体的构建体中观察到的能量转移量明显大于从各个供体到受体转移速率的总和预测的 FRET 效率。
结论/意义：我们得出的结论是，当存在多个受体时，要么存在额外的能量转移途径，要么动力学模型预测所基于的理论假设是不正确的。
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