4-fluorophenyl-L-alanine

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).

19F-NMR study of glucagon solvent accessibility at discrete sequence locations

The two phenylalanine residues in positions 6 and 22 of the glucagon amino acid sequence were substituted by 4F-Phe (Fig. a) in the recombinant variant polypeptide (Fig. b) by incubating E. coli cells with an excess of 4F-Phe, which was then incorporated via the naturally occurring tRNAPhe. 19F-NMR spectra were obtained for 4F-Phe and for 4F-Phe-labeled glucagon in H2O/D2O solutions with variable D2O concentrations (Fig. a, c). Samples of 4 mM 4F-Phe and 0.1 mM 4F-Phe-labeled glucagon in acidic water (pH* 2.5) were prepared with addition of D2O at 10% increments for 19F-NMR spectra collection. Within the NMR tube, a capillary containing 5 mM trifluoroacetic acid (TFA) was used as an external standard, and 19F chemical shift values were referenced to TFA at −76.55 ppm. Except for the spectra shown in Fig. b (see the figure caption for details), the 19F-NMR experiments were performed at 298 K on a Bruker AVANCE-500 spectrometer equipped with a room temperature BBO probehead (Bruker Biospin), using the pulse program zgfhigqn30.2 with 1H-decoupling during 19F-detection. The 19F frequency was about 470 MHz, and the number of scans was 128 for 4F-Phe, and 2048 for 4F-Phe-labeled glucagon. Prior to Fourier transformation, the time-domain NMR data were multiplied with an exponential function with line broadening factors of 1 and 5 Hz for 4F-Phe and 4F-Phe-labeled glucagon, respectively.

19F-NMR study of glucagon solvent accessibility at discrete sequence locations

Just as with 4F-Phe, the 19F chemical shift values of both labels in glucagon decreased linearly with increasing D2O concentration (Fig. d). The maximum 19F chemical shift differences were 0.14 ppm for 4F-Phe6 and 0.08 ppm for 4F-Phe22. These differences suggest that position 6 has full solvent exposure, whereas position 22 is only partially exposed to solvent. This result is readily rationalized by structural data from previous 1H-NMR studies of glucagon in acidic aqueous solutions, which showed that the region around residue six is flexibly disordered and Phe22 is part of a hydrophobic cluster formed by the residues –Phe22–Val23–Gln24–Trp25– (Boesch et al. ).

Introduction

Here, glucagon was labeled by substituting Phe in positions 6 and 22 with the non-proteinogenic amino acid 4-fluoro-l-phenylalanine (4F-Phe) (Bann and Frieden ; Li and Frieden ). The solvent isotope effect on the 19F chemical shift at variable D2O concentrations (Hansen et al. ; Kitevski-LeBlanc et al. ; Shi et al. ) was used to investigate site-specific solvent accessibility of glucagon (Fig. ). As a control, 1D 1H-NMR and cAMP assays were used to show that 4F-Phe incorporation caused at most minimal perturbations of glucagon conformation and activity (Fig. ). Our work lays a foundation for studying glucagon conformation and dynamics when interacting with GCGR with and without the presence of intramolecular partner proteins. Further investigation of glucagon–GCGR interactions in solution is particularly important given recent crystallographic evidence that an apparent small-molecule antagonist actually binds as an allosteric effector to the intracellular side of GCGR at the distance of ~20 Å from the orthosteric ligand binding site (Jazayeri et al. ).

Glucagon conformation and biological activity are preserved after 4F-Phe incorporation

Glucagon activates adenylate cyclase by binding to GCGR and activating G proteins (Bataille and Dalle ). We applied cAMP assays (Norskov-Lauritsen et al. ) to test [4F-Phe6,22]-glucagon for activity. The binding curves demonstrated that [4F-Phe6,22]-glucagon had similar activity as glucagon (Fig. g), indicating that this 19F-labeling does not affect glucagon signaling activity.

2. Results and Discussion

The toxicity of BMAA in comparison to l-4-fluorophenylalanine (FPA) and m-tyr was determined by evaluating metabolic activity and apoptosis/necrosis in three human cell lines, so as to determine whether BMAA produced the same type of toxicity at similar concentrations as the known misincorporating amino acid analogues. The rationale was that if BMAA did not produce any toxicity, or produced a different profile of apoptosis and necrosis with increasing concentrations in any of the cell lines, the mechanism of toxicity must differ. Amino acid ratios, including the ratio of the analogue to the replaced encoded amino acid, of SDS-PAGE purified proteins from exposed cell lines were then used to determine whether any toxicity observed was the result of misincorporation of the analogue.

2.1. Metabolic Activity

shows the activity, as measured by the metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), of FPA, m-tyr, and BMAA-exposed HepG2 (A), Caco-2 (B) and HeLa (C) cells at analogue concentrations ranging from 0.25 mM to 2 mM. FPA decreased metabolic activity at all the concentrations tested in all cell lines. m-Tyr decreased metabolic activity in both Caco-2 and HeLa cell lines at all tested concentrations, but only at the highest tested concentration in HepG2 cells. BMAA had no significant effect on HepG2 or HeLa cells at any of the tested concentrations. Interestingly, in Caco-2 cells, BMAA at 1 mM and 2 mM significantly enhanced the metabolic activity. These data indicate an absence of BMAA toxicity in these non-neuronal cell lines.

2.2. Apoptosis/Necrosis

A concentration-dependent decrease in total cell numbers was observed for both HepG2 (A) and Caco-2 (A) cells exposed to m-tyr and FPA. A slight but significant decrease in cell number was observed for HepG2 cells exposed to BMAA at the highest tested concentrations (2 mM) but BMAA did not affect cell growth and division at any of the tested concentrations in Caco-2 cells (). The different response of cells to BMAA, compared to known misincorporating amino acid analogues, suggests that BMAA, where toxic at the highest concentration for the one cell line, might have a different mechanism of toxicity. This is supported by the absence of an effect by BMAA on MTT reduction.

2.3.2. l-4-Fluorophenylalanine Analysis

As for m-tyr, there is a clear and steady increase in the FPA/l-phenylalanine ratios with increasing concentration of FPA as shown in . Again, there is also a trend of increasing FPA/l-serine and FPA/l-glycine concentrations with increasing FPA exposure concentrations, as well as decreasing l-phenylalanine/l-serine and l-phenylalanine/l-glycine ratios as more l-phenylalanine is replaced with higher concentrations of FPA.