<scp>FRETing</scp> about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance‐dependent energy transfer

Cory, Michael; Jones, Chloe; Shaffer, Kyle D; Venkatesh, Yarra; Giannakoulias, Sam; Perez, Ryann M.; Lougee, Marshall G.; Hummingbird, Eshe; Pagar, Vinayak Vishnu; Hurley, Christina M; Li, Allen; Mach, Robert H.; Kohli, Rahul M.; Petersson, E. James

doi:10.1002/pro.4633

Cited by 5 publications

(1 citation statement)

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“…The agreement between our measured FRET efficiencies and predicted distance distributions (Figure 8A) is encouraging. However, there are several limitations to FRET that reduce our present ability to extract structural or energetic information from tmFRET data (21). 1) The position of the donor fluorophore and acceptor metal ion may not faithfully report the position of the protein backbone, a problem we have partially mitigated with the use of small donors and acceptors with short linkers to the backbone.…”

Section: Discussionmentioning

confidence: 99%

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands

Gordon,

Evans,

Otto

et al. 2023

Preprint

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With the great progress on determining protein structures over the last decade comes a renewed appreciation that structures must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET) provides a great window into dynamics and energetics due to its application at physiological temperatures and ability to measure dynamics on the ångström scale. We have recently advanced transition metal FRET (tmFRET) to study allosteric regulation of maltose binding protein and have reported measurements of maltose-dependent distance changes with an accuracy of ~1.5 Å. When paired with the noncanonical amino acid Acd as a donor, our previous tmFRET acceptors were useful over a working distance of 10 Å to 20 Å. Here, we use cysteine-reactive bipyridyl and phenanthroline compounds as chelators for novel Fe2+- and Ru2+-based tmFRET acceptors to expand the working distance to as long as 50 Å, while preserving our ability to resolve even small maltose-dependent changes in distance. We compare our measured FRET efficiencies to predictions based on models using rotameric ensembles of the donors and acceptors to demonstrate that steady-state measurements of tmFRET with our new probes have unprecedented ability to measure conformational rearrangements under physiological conditions.

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Section: Discussionmentioning

confidence: 99%

Long-distance tmFRET using bipyridyl- and phenanthroline-based ligands

Gordon,

Evans,

Otto

et al. 2023

Preprint

Self Cite

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FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance‐dependent energy transfer

et al. 2023

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Förster resonance energy transfer (FRET) is a valuable method for monitoring protein conformation and biomolecular interactions. Intrinsically fluorescent amino acids that can be genetically encoded, such as acridonylalanine (Acd), are particularly useful for FRET studies. However, quantitative interpretation of FRET data to derive distance information requires careful use of controls and consideration of photophysical effects. Here we present two case studies illustrating how Acd can be used in FRET experiments to study small molecule induced conformational changes and multicomponent biomolecular complexes.

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