Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET.

Publisher: Cell Press Country of Publication: United States NLM ID: 0370626 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1542-0086 (Electronic) Linking ISSN: 00063495 NLM ISO Abbreviation: Biophys J Subsets: MEDLINE

Publication: Cambridge, MA : Cell Press
Original Publication: New York, Published by Rockefeller University Press [etc.] for the Biophysical Society.

Fluorescence Resonance Energy Transfer* ; Protein Conformation* ; Maltose-Binding Proteins*/chemistry ; Maltose-Binding Proteins*/metabolism; Allosteric Regulation ; Time Factors

Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose-binding protein, a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing that Cu(phen) ²⁺ can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method that also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen) ²⁺ as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.
Competing Interests: Declaration of interests The authors declare no competing interests.
(Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Update of: bioRxiv. 2024 Jan 03:2023.10.09.561594. doi: 10.1101/2023.10.09.561594. (PMID: 37873384)

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R01 EY010329 United States EY NEI NIH HHS; R01 GM125753 United States GM NIGMS NIH HHS; R35 GM145225 United States GM NIGMS NIH HHS; R35 GM148137 United States GM NIGMS NIH HHS; R01 EY017564 United States EY NEI NIH HHS; R03 TR004135 United States TR NCATS NIH HHS; T32 EY007031 United States EY NEI NIH HHS; T32 GM008268 United States GM NIGMS NIH HHS

0 (Maltose-Binding Proteins)

Date Created: 20240202 Date Completed: 20240717 Latest Revision: 20241118

20241119

PMC11309986

10.1016/j.bpj.2024.01.033

38303511