Using Fluorine Nuclear Magnetic Resonance To Probe the Interaction of Membrane-Active Peptides with the Lipid Bilayer

Abstract

A variety of biologically active peptides exert their function through direct interactions with the lipid membrane of the cell. These surface interactions are generally transient and highly dynamic, making them hard to study. Here we have examined the feasibility of using solution phase (19)F nuclear magnetic resonance (NMR) to study peptide-membrane interactions. Using the antimicrobial peptide MSI-78 as a model system, we demonstrate that peptide binding to either small unilamellar vesicles (SUVs) or bicelles can readily be detected by simple one-dimensional (19)F NMR experiments with peptides labeled with l-4,4,4-trifluoroethylglycine. The (19)F chemical shift associated with the peptide-membrane complex is sensitive both to the position of the trifluoromethyl reporter group (whether in the hydrophobic face or positively charged face of the amphipathic peptide) and to the curvature of the lipid bilayer (whether the peptide is bound to SUVs or bicelles). (19)F spin echo experiments using the Carr-Purcell-Meiboom-Gill pulse sequence were used to measure the transverse relaxation (T(2)) of the nucleus and thereby examine the local mobility of the MSI-78 analogues bound to bicelles. The fluorine probe positioned in the hydrophobic face of the peptide relaxes at a rate that correlates with the tumbling of the bicelle, suggesting that it is relatively immobile, whereas the probe at the positively charged face relaxes more slowly, indicating this position is much more dynamic. These results are in accord with structural models of MSI-78 bound to lipids and point to the feasibility of using fluorine-labeled peptides to monitor peptide-membrane interactions in living cells.

19F NMR Reveals Multiple Conformations at the Dimer Interface of the Nonstructural Protein 1 Effector Domain from Influenza A Virus

“…In spite of evidence for some exchange broadening of the Trp187 side chain H ε 1 resonance compared to the analogous resonances for other tryptophan residues in NS1A ED (Figure 1C), this 1 H resonance features a much narrower chemical shift dispersion and a different local environment, rendering it less sensitive to conformational exchange phenomena compared to the fluorinated tryptophan strategy. In the context of the broader 19 F protein NMR literature, these dynamical results on the NS1A ED dimer interface constitute a novel application of 19 F NMR relaxation to directly probe slow motion dynamics within a protein:protein interface The NMR properties of 19 F have, have been exploited recently to illuminate slow motion conformational dynamic processes in other types of interactions including protein-ligand interactions (Ahmed et al, 2007), peptide-bicelleinteractions (Buer et al, 2010), changes in dynamics accompanying protein complex formation (Acchione et al, 2012), and the thermodynamic and kinetic aspects of domain swapping (Liu et al, 2012). In addition, 19 F CPMG relaxation approaches were recently employed to characterize the conformational exchange behavior of fluorinated Phe residues in the thermal folding intermediate of calmodulin (Kitevski-Leblanc et al, 2013).…”

Effects of Peptide Immobilization Sites on the Structure and Activity of Surface-Tethered Antimicrobial Peptides

“…It has been reported that MSI-78 adopts an α-helical structure on association with the cell membrane and a random coil structure in aqueous buffer solutions. 27,28 Its short sequence and α-helical structure make it a good choice for structural characterization.…”

Structural basis for the enhanced stability of highly fluorinated proteins

“…The introduction of fluorinated amino acids into proteins has attracted particular interest, because although essentially absent from biology, fluorine has proved a remarkably useful element to probe the workings of biological molecules. For example, fluorinated substrates have been extensively used to investigate enzyme mechanisms, and 19 F NMR has proved a valuable tool for studying structure, dynamics, and interactions of fluorine-labeled proteins, peptides, lipids, and nucleic acids (4)(5)(6)(7)(8)(9). Fluorinated molecules also have important medical applications, exemplified by 20% of all pharmaceuticals containing fluorine, which improves pharmacokinetic properties (10).…”