TOAC spin-labeled peptides tailored for DNP-NMR studies in lipid membrane environments

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was similar to 1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better C-13 and N-15 signal NMR enhancements and H-1 spin-lattice T-1 relaxation than T-MacW.

Automated summary

The combination of in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures allows for the delivery of a radical source into cells via nitroxide-tagged peptide, enabling in situ biophysical studies of antimicrobial peptides. This study investigated the structure, localization, and signal enhancement properties of spin-labeled Mac1 analogs within micelles or lipid vesicles, with the double spin-labeled peptide achieving better NMR enhancements. Molecular dynamics simulations supported the observations and provided further structural insights.