4‐Fluorophenylglycine as a Label for ¹⁹F NMR Structure Analysis of Membrane‐Associated Peptides

Abstract

The non-natural amino acid 4-fluorophenylglycine (4F-Phg) was incorporated into several representative membrane-associated peptides for dual purpose. The (19)F-substituted ring is directly attached to the peptide backbone, so it not only provides a well-defined label for highly sensitive (19)F NMR studies but, in addition, the D and L enantiomers of the stiff side chain may serve as reporter groups on the transient peptide conformation during the biological function. Besides peptide synthesis, which is accompanied by racemisation of 4F-Phg, we also describe separation of the epimers by HPLC and removal of trifluoroacetic acid. As a first example, 18 different analogues of the fusogenic peptide "B18" were prepared and tested for induction of vesicle fusion; the results confirmed that hydrophobic sites tolerated 4F-Phg labelling. Similar fusion activities within each pair of epimers suggest that the peptide is less structured in the fusogenic transition state than in the helical ground state. In a second example, five doubly labelled analogues of the antimicrobial peptide gramicidin S were compared by using bacterial growth inhibition assays. This cyclic beta-sheet peptide could accommodate both L and D substituents on its hydrophobic face. As a third example, we tested six analogues of the antimicrobial peptide PGLa. The presence of d-4F-Phg reduced the biological activity of the peptide by interfering with its amphiphilic alpha-helical fold. Finally, to illustrate the numerous uses of l-4F-Phg in (19)F NMR spectroscopy, we characterised the interaction of labelled PGLa with uncharged and negatively charged membranes. Observing the signal of the free peptide in an aqueous suspension of unilamellar vesicles, we found a linear saturation behaviour that was dominated by electrostatic attraction of the cationic PGLa. Once the peptide is bound to the membrane, however, solid-state (19)F NMR spectroscopy of macroscopically oriented samples revealed that the charge density has virtually no further influence on the structure, alignment or mobility of the peptide.

Conformational Flexibility and Strand Arrangements of the Membrane-Associated HIV Fusion Peptide Trimer Probed by Solid-State NMR Spectroscopy

“…Other investigators have proposed that the peptide structure responsible for fusion is irregular and may be transient (107,108). Although the present paper does not directly address these issues, it is noted: (1) cholesterol-associated structural variation is also observed for the influenza virus fusion peptide (27,76); and (2) pH-triggered fusion can be observed both for helical influenza fusion peptide bound to non-cholesterol containing membranes and for β strand influenza fusion peptide bound to cholesterol-containing membranes (109).…”

NMR methods for studying membrane‐active antimicrobial peptides

“…Deuterium labels have been incorporated uniformly, and in many cases also specifically at the H ␣ position. A number of aromatic and aliphatic amino acids with a 19 F label in the side chain are available but may be difficult to purchase as pure L-enantiomers (64,65).…”

“…Several antimicrobial peptides have been studied by 19 F NMR in lipid bilayers, labeled with either 4F-phenylglycine (4F-Phg), 4-CF 3 -phenylglycine (CF 3 -Phg), or 3F-alanine (F-Ala) (64,65). In all these unnatural amino acids, the 19 F reporter group is directly attached to the C ␣ atom, thus forming a rigid connection to the peptide backbone.…”

Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide to Lipid Distances Reveal the Intimate Contact of β Strand Peptide with Membranes and the Proximity of the Ala-14−Gly-16 Region with Lipid Headgroups

“…Oligomerization/aggregation has also been detected by other biophysical methods (15,30). There is evidence that at least the lipid mixing step of membrane fusion can occur with the HFP in either helical or β strand conformation although there is some controversy in the literature about this conclusion (14,16,18,(31)(32)(33)(34)(35).HFP location in membranes has been primarily probed using a HFP-F8W mutant and by variation of the tryptophan fluorescence of this mutant with changes in environment (36,37). Key results have included: (1) fluorescence was higher for membrane-associated HFP-F8W than for HFP-F8W in buffered saline solution; (2) greater fluorescence quenching by acrylamide was observed for a soluble tryptophan analog than for membrane-associated HFP-F8W; and (3) similar fluorescence quenching of membrane-associated HFP-F8W was observed in samples containing either 1-palmitoyl-2-stearoyl-phosphocholine brominated at the 6, 7 carbons of the stearoyl chain or the corresponding lipid brominated at the 11, 12 carbons of the chain.…”