Anomalous surplus energy transfer observed with multiple FRET acceptors.
Anomalous surplus energy transfer observed with multiple FRET acceptors.
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DOI：
10.1371/journal.pone.0008031
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发表时间：
2009-11-25
期刊：
影响因子：
3.7
通讯作者：
Vogel SS
中科院分区：
文献类型：
--
作者：
Koushik SV;Blank PS;Vogel SS
Förster共振能量转移(FRET)是一种通过非辐射偶极-偶极相互作用将能量从被激发的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-受体对之间的相互作用。不幸的是，人们很少知道在一个分子复合体中是否只有一个受体。因此，使用FRET作为一种工具来测量活细胞内的蛋白质-蛋白质相互作用，需要了解FRET如何随着多个受体的变化而变化。当存在多个FRET受体时，假设一个量子的能量要么从供体释放，要么全部转移到只有一个存在的受体。在没有其他受体的情况下，可以测量施主和特定受体之间的能量转移速率(Kd→A)，并且这些单独的FRET转移速率可以用一个简单的动力学模型来预测整体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。通过测量两个结构中的整体FRET效率来测试这种方法的一般性，每个结构都包含一个荧光蛋白供体(Cerulean)和两个或三个FRET受体(Venus)。在系统地引入点突变以消除所有其他受体后，根据系统地引入点突变以消除所有其他受体后测量的FRET效率来计算这些结构中的单个供体-受体对之间的FRET转移率。我们发现，在具有多个受体的结构中观察到的能量转移量显著大于从单个供体到受体转移率之和预测的FRET效率。我们的结论是，当存在多个受体时，要么存在额外的能量转移途径，要么是动力学模型预测所基于的理论假设是不正确的。
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 (kD→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. 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. 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.
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影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
DOI：
10.1073/pnas.251525598
发表时间：
2001-12-04
影响因子：
11.1
作者：
Garcia-Parajo, MF;Koopman, M;van Hulst, NF
通讯作者：
van Hulst, NF
影响因子：
3.3
作者：
Lounis, B;Deich, J;Moerner, WE
通讯作者：
Moerner, WE
影响因子：
2.4
作者：
FORSTER, T
通讯作者：
FORSTER, T
DOI：
10.1073/pnas.97.22.11996
发表时间：
2000-10-24
影响因子：
11.1
作者：
Heikal, AA;Hess, ST;Webb, WW
通讯作者：
Webb, WW