Solvent-accessibility of discrete residue positions in the polypeptide hormone glucagon by 19F-NMR observation of 4-fluorophenylalanine

Abstract

The amino acid 4-fluoro-l-phenylalanine (4F-Phe) was introduced at the positions of Phe6 and Phe22 in the 29-residue polypeptide hormone glucagon by expressing glucagon in E. coli in the presence of an excess of 4F-Phe. Glucagon regulates blood glucose homeostasis by interaction with the glucagon receptor (GCGR), a class B GPCR. By referencing to the 4F-Phe chemical shifts at varying D2O concentrations, the solvent exposure of the two Phe sites along the glucagon sequence was determined, showing that 4F-Phe6 was fully solvent exposed and 4F-Phe22 was only partially exposed. The incorporation of fluorine atoms in polypeptide hormones paves the way for novel studies of their interactions with membrane-spanning receptors, specifically by differentiating between effects on the solvent accessibility, the line shapes, and the chemical shifts from interactions with lipids, detergents and proteins. Studies of interactions of GCGR with ligands in solution is at this point of keen interest, given that recent crystallographic studies revealed that an apparent small molecule antagonist actually binds as an allosteric effector at a distance of ~20 Å from the orthosteric ligand binding site (Jazayeri et al., in Nature 533:274–277, 2016).

GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures

“…Class B GPCRs offer new possibilities for applying NMR to study mechanisms of ligand recognition and intracellular signaling. Most endogenous Class B GPCR ligands are polypeptides, which can be labeled with stable isotopes 187 to study their detailed atomic interactions with receptors by NMR. The possibility also arises to use NMR in parallel with intein chemistry to selectively label different segments of the GPCR sequence with 2 H, 15 N and/or 13 C. Intein chemistry has been applied to study a C-terminal polypeptide segment of β 2 AR 124 , and studies of selectively labeled Class B receptor extracellular domains within the full-length receptor would be an obvious extension of this work.…”

(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5′-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect

“…The 19 F chemical shifts of fluorobenzenes are known to be sensitive to isotope effects, and their sensitivity to D 2 O versus H 2 O solvent has been used to identify solvent accessibility of fluorinated aromatic amino acids. − The isotope effects observed for the difluoroleucine residues in PpiB-d were smaller and did not reflect solvent exposure in a straightforward manner. The largest effect was observed for the low-field 19 F NMR signal of the highly solvent-exposed residue 115, which was more shielded by 0.19 ppm in 80% D 2 O/20% H 2 O versus 90% H 2 O/10% D 2 O.…”

GPCR structural characterization by NMR spectroscopy in solution

“…Furthermore, 19 F is rarely found in biological macromolecules, which enables the NMR detection of extrinsic 19 F labels with minimal background signals. 19 F-probes can be introduced by post-translational chemical modification [30], and they have been widely used for studies of structure and dynamics of proteins during the past several decades [31][32][33][34]. As with all extrinsic probes, one has to deal with the concern that fluorine labeling could affect the conformation of the protein.…”