2D 1H/1H RFDR and NOESY NMR experiments on a membrane-bound antimicrobial peptide under magic angle spinning.

There is significant interest in solving high-resolution dynamic structures of membrane-associated peptides using solid-state NMR spectroscopy. Previous solid-state NMR studies have provided valuable insights into the functional properties of an exciting class of biomacromolecules such as antimicrobial peptides and amyloid peptides. However, it has been a major challenge to apply solid-state NMR techniques to study peptides or proteins that are not labeled with specific isotopes such as (13)C, (15)N, and/or (2)H. This study utilizes 2D (1)H/(1)H radio frequency-driven recoupling (RFDR) and nuclear Overhauser effect spectroscopy (NOESY) pulse sequences under magic angle spinning to study a membrane-bound antimicrobial peptide MSI-78 (or also known as pexiganan). We demonstrate that proton resonances can be assigned and structural constraints, NOE and (1)H-(1)H dipolar couplings, can be measured without the need for any isotopic enrichment. The buildup curves, showing the dependence of the cross peak intensity against the mixing time, obtained from 2D (1)H/(1)H NOESY and RFDR experiments are compared. Our results reveal that the RFDR-recovered (1)H-(1)H dipolar couplings associated with alpha and side chain protons are larger than that with the amide-protons. This study provides a means to measure residual (1)H-(1)H dipolar couplings for the investigation of structure, dynamics, and aggregation of peptides using a suitable model membrane like micelles or bicelles.