Estimating the distance separating fluorescent protein FRET pairs.
Estimating the distance separating fluorescent protein FRET pairs.
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DOI：
10.1016/j.ymeth.2013.06.021
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发表时间：
2014-03-15
期刊：
影响因子：
4.8
通讯作者：
Blank, Paul S.
中科院分区：
文献类型：
--
作者：
Vogel, Steven S.;van der Meer, B. Wieb;Blank, Paul S.
关键词：
Förster共振能量转移（FRET）描述了一种广泛应用于生物医学研究的物理现象，用于估计生物分子之间的分离。此外，基因工程用于将绿色荧光蛋白（GFP）的光谱变体掺入细胞表达的蛋白质中。然后测定供体和受体FP之间的能量转移效率或速率。由于仅当供体和受体非常接近（1-10 nm）时才发生明显的FRET，因此FRET的存在可能表明工程化蛋白质作为相互作用物质缔合。对于一个均匀的人口FRET对FRET供体和受体之间的分离，可以估计从测量的FRET效率，如果它是假设，供体和受体是随机取向和旋转广泛在其激发态（动态制度）。与典型的有机荧光团不同，FP的旋转相关时间通常比其荧光寿命长得多;因此FP在其激发态期间几乎是静态的。因此，估计FP FRET对之间的分离是有问题的。为了克服这一障碍，我们在这里提出了一个简单的方法来估计使用实验测得的平均FRET效率FP之间的分离。这种方法假设供体和受体荧光团是随机取向的，但在其激发态（静态状态）期间不旋转。这种方法利用蒙特-卡罗模拟生成的查找表，允许一个估计的分离，归一化到Förster距离，从平均FRET效率。与假设静态状态相比，假设动态状态会显著高估分离（效率在0.5附近高估10%，在0.75附近高估30%），静态状态更适合于FP之间的分离估计。
Förster resonance energy transfer (FRET) describes a physical phenomenon widely applied in biomedical research to estimate separations between biological molecules. Routinely, genetic engineering is used to incorporate spectral variants of the green fluorescent protein (GFPs), into cellular expressed proteins. The transfer efficiency or rate of energy transfer between donor and acceptor FPs is then assayed. As appreciable FRET occurs only when donors and acceptors are in close proximity (1–10 nm), the presence of FRET may indicate that the engineered proteins associate as interacting species. For a homogeneous population of FRET pairs the separations between FRET donors and acceptors can be estimated from a measured FRET efficiency if it is assumed that donors and acceptors are randomly oriented and rotate extensively during their excited state (dynamic regime). Unlike typical organic fluorophores, the rotational correlation-times of FPs are typically much longer than their fluorescence lifetime; accordingly FPs are virtually static during their excited state. Thus, estimating separations between FP FRET pairs is problematic. To overcome this obstacle, we present here a simple method for estimating separations between FPs using the experimentally measured average FRET efficiency. This approach assumes that donor and acceptor fluorophores are randomly oriented, but do not rotate during their excited state (static regime). This approach utilizes a Monte-Carlo simulation generated look-up table that allows one to estimate the separation, normalized to the Förster distance, from the average FRET efficiency. Assuming a dynamic regime overestimates the separation significantly (by 10% near 0.5 and 30% near 0.75 efficiencies) compared to assuming a static regime, which is more appropriate for estimates of separations between FPs.
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