Available online 21 August 2023, 102377

Author links open overlay panel, Abstract

19F NMR provides a way of monitoring conformational dynamics of G-protein coupled receptors (GPCRs) from the perspective of an ensemble. While X-ray crystallography provides exquisitely resolved high-resolution structures of specific states, it generally does not recapitulate the true ensemble of functional states. Fluorine (19F) NMR provides a highly sensitive spectroscopic window into the conformational ensemble, generally permitting the direct quantification of resolvable states. Moreover, straightforward T1- and T2-based relaxation experiments allow for the study of fluctuations within a given state and exchange between states, on timescales spanning nanoseconds to seconds. Conveniently, most biological systems are free of fluorine. Thus, via fluorinated amino acid analogues or thiol-reactive fluorinated tags, F or CF3 reporters can be site specifically incorporated into proteins of interest. In this review, fluorine labeling protocols and 19F NMR experiments will be presented, from the perspective of small molecule NMR (i.e. drug or small molecule interactions with receptors) or macromolecular NMR (i.e. conformational dynamics of receptors and receptor–G-protein complexes).

Section snippetsIntroduction and overview of NMR approaches to the study of GPCRs

The G Protein-Coupled Receptor (GPCR) superfamily consists of over 830 distinct 7-TM proteins, governing sensory and neuronal signaling, cell homeostasis, and immune response. Over 360 of these receptors are endo-GPCRs (other than olfactory, taste, and visual) and are thus potential drug targets, either via their orthosteric binding sites or via a variety of potential allosteric hot spots [1∗, 2, 3]. The diverse structures observed in and around the orthosteric pocket of the GPCR superfamily

Labeling: Heterologous expression, cell-free expression, and unnatural amino acids (UAAs)

Fluorinated reporters can be incorporated via heterologous or cell-free expression systems, using either fluorinated amino acid analogues that are tolerated by the natural amino acid tRNA synthetase, or fluorinated unnatural amino acids, which utilize AMBER stop codon technology [32]. Fluorinated mimics of aliphatic residues (leucine, isoleucine, valine, alanine, proline, and methionine) and aromatic residues (tryptophan, tyrosine, and phenylalanine) can be used in heterologous expression

Conformational dynamics

In keeping with their pharmacology, the conformational ensemble of GPCRs spans distinct inactive states, pre-activated states that facilitate G protein binding, and specific activation intermediates, which enable dynamics and G protein coupling (i.e. nucleotide exchange and functional dissociation of the G protein) [13,17,50∗, 51, 52, 53∗, 54, 55, 56, 57]. In many cases, these functional states can be simultaneously detected by 19F NMR. The addition of allosteric adjuvants, receptor ligands,

Future applications and conclusions

Modern drug discovery relies heavily on ultra-large virtual libraries and extensive docking studies of candidate molecules. As a result, it is much easier to initiate an experimental drug discovery project using 19F-labeled fragments which are anticipated to bind to specific regions. CPMG-filtered 19F NMR spectroscopy could in this case be used in conjunction with spy lead compounds or exclusively with fluorinated fragment libraries. The availability of high sensitivity and automated NMR

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Canadian Institutes of Health Research (CIHR) Operating Grant PJT-183778 to R.S.P.

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