Premium
Nitroxide spin–labeled peptides for DNP‐NMR in‐cell studies
Author(s) -
Sani Marc-Antoine,
Zhu Shiying,
Hofferek Vinzenz,
Separovic Frances
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.201900931r
Subject(s) - nitroxide mediated radical polymerization , chemistry , magainin , membrane , nuclear magnetic resonance spectroscopy , cell , biophysics , crystallography , peptide , nuclear magnetic resonance , stereochemistry , antimicrobial peptides , biochemistry , biology , organic chemistry , physics , radical polymerization , copolymer , polymer
Antimicrobial peptides (AMPs) that target lipid membranes show promise as alternatives to conventional antibiotics. However, the molecular mechanisms of membrane perturbation, as most studies are performed in model systems and in‐cell structural studies, have yet to be achieved. Solid‐state NMR spectroscopy is a valuable technique to investigate peptide‐membrane interactions and to determine the structure of peptides, but the short lifespan of bacteria, especially under magic angle spinning conditions, has not permitted in‐cell structural studies. Here, we present the first dynamic nuclear polarization (DNP)‐NMR in‐cell studies of Escherichia coli bacteria incubated with the AMP maculatin 1.1 (Mac1) in combination with novel nitroxide spin‐labeled peptides 2,2,6,6‐tetramethylpi‐peridine‐N‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC)–[F3W]‐Mac1 (MacW) and TOAC‐TOAC‐MacW. The in‐cell 13 C and 15 N signal NMR enhancements, and 1 H spin‐lattice T 1 relaxation times showed that TOAC‐MacW and TOAC‐TOAC‐MacW performed better than the more hydrophilic biradical AMUPol used for DNP studies. Furthermore, the pores formed by the AMP increased the signal enhancements and decreased T 1 values of specifically 13 C‐ and 15 N‐labeled Mac1. This approach has a great potential for determining the first in situ structures of AMPs in bacteria.—Sani, M.‐A., Zhu, S., Hofferek, V., Separovic, F. Nitroxide spin–labeled peptides for DNP‐NMR in‐cell studies. FASEB J. 33, 11021–11027 (2019). www.fasebj.org