Effect of enhanced Renilla luciferase and fluorescent protein variants on the Forster distance of Bioluminescence resonance energy transfer (BRET)
Effect of enhanced Renilla luciferase and fluorescent protein variants on the Forster distance of Bioluminescence resonance energy transfer (BRET)
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DOI：
10.1016/j.bbrc.2012.07.133
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发表时间：
2012-08-31
影响因子：
3.1
通讯作者：
Trowell, Stephen C.
中科院分区：
文献类型：
--
作者：
Dacres, Helen;Michie, Michelle;Trowell, Stephen C.
生物发光共振能量转移（BRET）是监测大分子相互作用的重要工具，也是生物传感器开发中的一种有用的转导技术。福斯特距离（R-0），分子间的分离，其特征在于50%的最大可能的能量转移，是一个关键的BRET参数。R-0提供了一种将BRET比率的测量变化与物理尺寸尺度联系起来的方法，并允许估计任何供体-受体对可以测量的距离范围。BRET测定的灵敏度最近已经通过引入新的BRET组分RLuc 2、RLuc 8和Venus而得到改善，其具有改善的量子产率、稳定性和亮度。我们确定了BRET 1系统的R-0，包括新的RLuc变体RLuc 2或RLuc 8，与Venus的组合，分别为5.68或5.55 nm。这些值比原始BRET 1系统的R-0高约25%。将绿色荧光蛋白（GFP（2））与RLuc 2或RLuc 8变体组合的BRET 2系统的R-0为7.67或8.15 nm，即仅比原始BRET 2系统大2-9%，尽管类似于30倍更亮。皇冠版权所有（C）2012由Elsevier Inc.发布。All rights reserved.
Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Forster distance (R-0), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R-0 provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor-acceptor pair. The sensitivity of BRET assays has recently been improved by introduction of new BRET components, RLuc2, RLuc8 and Venus with improved quantum yields, stability and brightness. We determined R-0 for BRET1 systems incorporating novel RLuc variants RLuc2 or RLuc8, in combination with Venus, as 5.68 or 5.55 nm respectively. These values were approximately 25% higher than the R-0 of the original BRET1 system. R-0 for BRET2 systems combining green fluorescent proteins (GFP(2)) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2-9% greater than the original BRET2 system despite being similar to 30-fold brighter. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved.