Abstract
ABSTRACTWith 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 Fe2+and Ru2+to produce novel 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.STATEMENT OF SIGNIFICANCEIn this work, we expand the working distance of transition metal ion Förster Resonance Energy Transfer (tmFRET) to allow the measurement of donor-acceptor distances from 10 Å to 50 Å. We develop new cysteine-reactive bipyridyl- and phenanthroline-based ligands for Cu2+, Fe2+, and Ru2+and examine their ability to resolve small ligand-dependent distance changes in maltose binding protein. We extend these studies using pulsed electron paramagnetic resonance spectroscopy to demonstrate the high accuracy of tmFRET for studying protein allostery.
Publisher
Cold Spring Harbor Laboratory
Cited by
1 articles.
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