Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels

Abstract

To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on 13C- and/or 15N-enrichment alongside 1H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of 19F nuclei into a sample. The 19F nucleus is highly sensitive to environmental changes and allows for one-dimensional NMR spectroscopic study, with perturbation to chemical shift and spin dynamics diagnostic of structural change, ligand binding, and modified conformational sampling. This was applied to the apelinergic system, which comprises a rhodopsin-like G protein-coupled receptor (the apelin receptor (AR)/APJ) and two families of cognate ligands, the apelin and apela (ELABELA/toddler) peptides. Specifically, AR fragments consisting of either the N-terminal tail and first transmembrane (TM) α-helix (AR55) or the first three transmembrane α-helices (TM1-3) were prepared with biosynthetic fluorotryptophan incorporation. Interactions of each AR fragment with a high-affinity, 2,4,5-trifluorophenylalanine labeled apelin analogue were compared by 19F NMR. Distinct ranges of 19F chemical shifts for ligand and receptor provide unambiguous tracking of both species, with distinct binding behaviour observed for each AR fragment implying that AR55 is not sufficient to recapitulate the physiological binding event. Site-specific perturbation was also apparent for the apelin analogue as a function of substitution site, indicating an orientational binding preference. As a whole, this strategy of distinctive 19F labelling for ligand and receptor provides a relatively fast (i.e., employing 1D NMR experiments) and highly sensitive method to simultaneously and definitively track binding in both species.

Nucleotide Binding and Active Site Gate Dynamics for the Hsp90 Chaperone ATPase Domain from Benchtop and High Field 19F NMR Spectroscopy

“…19 F NMR spectroscopy of biological systems has advantages of high sensitivity; the chemical shift is an exquisite reporter of subtle changes in chemical environment, and dynamic processes ranging from picoseconds to seconds can be characterized . Furthermore, given its near total absence in biological molecules, incorporation into large biomolecules and complexes facilitates versatile and affordable biophysical studies of such challenging systems. ,, …”

“…19 Furthermore, given its near total absence in biological molecules, incorporation into large biomolecules and complexes facilitates versatile and affordable biophysical studies of such challenging systems. 17,20,21 We chemically modified the Hsp90 ATPase domain with bromo-1,1,1-trifluoroacetone to generate two cysteine−TFA derivative residues (CYF residues) bearing a terminal trifluoromethyl group. One residue was engineered at position 61, which resides on a rigid, solvent-exposed loop, and another at position 110, which resides at the end of the first helix of the active site gate, near its apex.…”

Structure and Function of Membrane Proteins

“…An article in this Special Issue by the group of Rainey [10] highlights another unique approach to examine membrane proteins, in this case studying ligand-receptor binding at the atomic level. In this unique study, they exploit the lack of natural fluorine in proteins.…”