High Resolution Heteronuclear Correlation NMR Spectroscopy of an Antimicrobial Peptide in Aligned Lipid Bilayers: Peptide−Water Interactions at the Water−Bilayer Interface
Riqiang Fu*†, Eric D. Gordon‡, Daniel J. Hibbard‡ and Myriam Cotten*§
High-resolution two-dimensional (2D) 1H−15N heteronuclear correlation (HETCOR) spectroscopy has been used to characterize the structure and dynamics of 15N-backbone labeled antimicrobial piscidin 1 (p1) oriented in “native-like” hydrated lipid bilayers. Piscidin belongs to a family of amphipatic cationic antimicrobial peptides, which are membrane-active and have broad spectrum antimicrobial activity on bacteria. When the 1H chemical shifts are encoded by the 1H−15N dipolar couplings, 2D dipolar-encoded HETCOR (i.e., de-HETCOR) solid-state NMR spectra yield high resolution 1H and 15N chemical shifts as well as 1H−15N heteronuclear dipolar couplings. Several advantages of HETCOR and de-HETCOR techniques that emerge from our investigations could facilitate the atomic-level investigations of structure−function relationships in membrane-active peptides and membrane-bound species. First, the de-HETCOR NMR spectrum of a ten-site 15N-labeled sample of p1 aligned in hydrated lipid bilayers can resolve resonances that are overlapped in the standard HETCOR spectrum. Second, the resolution in de-HETCOR spectra of p1 improves significantly at higher magnetic field due to an enhanced alignment that improves spectrum definition uniformly. Third, the HETCOR spectrum of 15N−K14 p1 oriented in hydrated lipid bilayers displays not only the expected crosscorrelation between the chemical shifts of bonded amide1H and 15N spins but also a cross peak between the 1H chemical shift from bulk water and the 15N chemical shift from the labeled amide nitrogen. This information provides new insights into the intermolecular interactions of an amphipathic antimicrobial peptide optimized to partition at the water-bilayer interface and may have implications at the biological level.