Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET

Abstract

ABSTRACTProteins 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 (MBP), 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 Cu(phen)2+can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method which 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.STATEMENT OF SIGNIFICANCEResponsible for the regulation of virtually all biological processes, allosteric proteins are fundamental to all life. To understand the mechanisms for their vital functions, we must determine the structure and energetics of these proteins under physiological conditions. In this work, we have developed a fluorescence method that allows for the simultaneous measurement of protein structure and energetics under physiological conditions. These studies pave the way for future advances in physiology and medicine.