¹⁹F NMR as a versatile tool to study membrane protein structure and dynamics

Abstract

To elucidate the structures and dynamics of membrane proteins, highly advanced biophysical methods have been developed that often require significant resources, both for sample preparation and experimental analyses. For very complex systems, such as membrane transporters, ion channels or G-protein coupled receptors (GPCRs), the incorporation of a single reporter at a select site can significantly simplify the observables and the measurement/analysis requirements. Here we present examples using 19F nuclear magnetic resonance (NMR) spectroscopy as a powerful, yet relatively straightforward tool to study (membrane) protein structure, dynamics and ligand interactions. We summarize methods to incorporate 19F labels into proteins and discuss the type of information that can be readily obtained for membrane proteins already from relatively simple NMR spectra with a focus on GPCRs as the membrane protein family most extensively studied by this technique. In the future, these approaches may be of particular interest also for many proteins that undergo complex functional dynamics and/or contain unstructured regions and thus are not amenable to X-ray crystallography or cryo electron microscopy (cryoEM) studies.

Surface hydrophobics mediate functional dimerization of CYP121A1 of Mycobacterium tuberculosis

“…In order to understand how the dimer interface of CYP121A1 affects the local structure at this site, 19 F-NMR was used as a means to monitor the FG loop in both the monomer and dimer of the enzyme. Fluorine nuclei are naturally NMR observable (spin of ½), are highly sensitive to changes in the electrochemical environment, and can be incorporated in a site-specific manner 20,21 . Moreover, the sensitivity of the fluorine nucleus is particularly useful as a structural probe for a dimer complex that, due to its size, is otherwise inaccessible by a 15 N labeling strategy.…”

19F-NMR reveals substrate specificity of CYP121A1 in Mycobacterium tuberculosis

“…The 19 F nucleus exhibits similar sensitivity for NMR detection to that of proton nuclei. Moreover, because 19 F is not naturally incorporated into proteins, 19 F labeling results in one-dimensional spectra that are greatly simplified by a reduction in spectral crowding ( 8 , 9 ).…”

Multidrug Resistance in Mammals and Fungi—From MDR to PDR: A Rocky Road from Atomic Structures to Transport Mechanisms

“…Thus, we need atomic structures reflecting more than a single conformation and possibly many transition states [ 352 ], as well as extensive validation by biochemistry and genetics, to validate their biological relevance and define catalytic cycles. Furthermore, there is an unmet need for more interdisciplinary collaborations that also engage alternative structural approaches like NMR [ 353 , 354 , 355 , 356 ] as well as complementary biophysical methods [ 265 , 357 , 358 , 359 ] to expand our mechanistic views of ABC transporters in all living kingdoms.…”