Weihs Felix, Wang Jian, Pfleger Kevin D G, Dacres Helen
CSIRO Health & Biosecurity, Parkville, 343 Royal Parade, Melbourne, VIC, 3030, Australia.
CSIRO Health & Biosecurity, Canberra, ACT, 2601, Australia.
Anal Chim Acta X. 2020 Sep 2;6:100059. doi: 10.1016/j.acax.2020.100059. eCollection 2020 Nov.
Bioluminescence Resonance Energy Transfer (BRET) is widely applied to study protein-protein interactions, as well as increasingly to monitor both ligand binding and molecular rearrangements. The Förster distance (R) describes the physical distance between the two chromophores at which 50% of the maximal energy transfer occurs and it depends on the choice of RET components. R can be experimentally determined using flexible peptide linkers of known lengths to separate the two chromophores. Knowledge of the R helps to inform on the choice of BRET system. For example, we have previously shown that BRET exhibits the largest R to date for any genetically encoded RET pair, which may be advantageous for investigating large macromolecular complexes if its issues of low and fast-decaying bioluminescence signal can be accommodated. In this study we have determined R for a range of bright and red-shifted BRET pairs, including NanoBRET with tetramethylrhodamine (TMR), non-chloro TOM (NCT), mCherry or Venus as acceptor, and BRET, a red-shifted BRET-like system. This study revealed R values of 6.15 nm and 6.94 nm for NanoBRET using TMR or NCT as acceptor ligands, respectively. R was 5.43 nm for NanoLuc-mCherry, 5.59 nm for NanoLuc-Venus and 5.47 nm for BRET. This extends the palette of available BRET Förster distances, to give researchers a better-informed choice when considering BRET systems and points towards NanoBRET with NCT as a good alternative to BRET as an analysis tool for large macromolecular complexes.
生物发光共振能量转移（BRET）被广泛应用于研究蛋白质 - 蛋白质相互作用，并且越来越多地用于监测配体结合和分子重排。福斯特距离（R）描述了两个发色团之间的物理距离，在该距离处发生最大能量转移的50%，它取决于RET组件的选择。R可以通过使用已知长度的柔性肽接头来分离两个发色团进行实验测定。了解R有助于指导BRET系统的选择。例如，我们之前已经表明，对于任何基因编码的RET对，BRET展现出了迄今为止最大的R，如果其低且快速衰减的生物发光信号问题能够得到解决，这对于研究大型大分子复合物可能是有利的。在本研究中，我们测定了一系列明亮且红移的BRET对的R值，包括以四甲基罗丹明（TMR）、非氯TOM（NCT）、mCherry或Venus作为受体的NanoBRET，以及一种红移的类BRET系统。这项研究分别揭示了使用TMR或NCT作为受体配体的NanoBRET的R值为6.15 nm和6.94 nm。NanoLuc - mCherry的R为5.43 nm，NanoLuc - Venus的R为5.59 nm，BRET的R为5.47 nm。这扩展了可用的BRET福斯特距离范围，使研究人员在考虑BRET系统时能够做出更明智的选择，并表明以NCT作为受体的NanoBRET是作为大型大分子复合物分析工具的BRET的一个很好的替代方案。
