Solid-State NMR Structure Analysis of the Short Multifunctional Peptide BP100 in Membranes

Abstract

The 11-residue peptide BP100 (KKLFKKILKYL-amide) is a short antimicrobial peptide that has been optimized against plant pathogens, and it is also able to act as a cell-penetrating agent. To address the conformation, orientation and dynamics of BP100 in the membrane-bound state, we have labeled all of the six hydrophobic amino acids individually with CF3-L-Bpg for solid-state 19F-NMR analysis, and a 15N-label was incorporated at Leu8. Circular dichroism (CD) analysis showed that the substitutions did not perturb the overall structure of the α-helical peptide, and the antimicrobial activity also remained unaffected. Using highly sensitive 19F-NMR, we found that the orientation of BP100 in macroscopically oriented DMPC/PG membranes remains unchanged as a function of peptide concentration over a wide range of peptide-to-lipid molar ratios from 1:10 to 1:3000, accessible only by fluorine NMR. The 19F-NMR data analysis was compatible with a large family of possible helix tilt angles, but these could by narrowed down by including peptide dynamics in the structure determination process. These results were complemented by 15N-NMR and oriented CD spectroscopy, both showing that the amphiphilic BP100 helix is oriented parallel to the membrane surface. In summary, our results show that the short BP100 molecule assumes a surface bound state under different concentration regimes and remains highly mobile. We suggest that at low peptide concentration BP100 may be able to permeate cellular membranes in a transient way, but at high concentration it perturbs the membrane via a carpet mechanism.