Nitroxide spin-labeled peptides for DNP-NMR in-cell studies

Area of Science:

• Biophysics
• Structural Biology
• Microbiology

Background:

• Antimicrobial peptides (AMPs) targeting lipid membranes are potential antibiotic alternatives.
• Understanding AMP molecular mechanisms in bacteria requires in-cell structural studies, which are currently limited.
• Solid-state NMR is valuable for peptide-membrane interactions but challenged by bacterial viability for in-cell studies.

Purpose of the Study:

• To present the first dynamic nuclear polarization (DNP)-NMR in-cell studies of antimicrobial peptides (AMPs) in *Escherichia coli*.
• To evaluate novel nitroxide spin-labeled peptides for their efficacy in DNP-NMR in-cell applications.
• To investigate the structural basis of AMP-membrane interactions within live bacterial cells.

Main Methods:

• Dynamic Nuclear Polarization (DNP)-NMR spectroscopy was employed for in-cell studies.
• Bacteria (*Escherichia coli*) were incubated with nitroxide spin-labeled antimicrobial peptides: maculatin 1.1 (Mac1), TOAC-[F3W]-Mac1 (MacW), and TOAC-TOAC-MacW.
• In-cell 13C and 15N signal NMR enhancements and 1H spin-lattice T1 relaxation times were measured.

Main Results:

• Novel spin-labeled peptides (TOAC-MacW, TOAC-TOAC-MacW) demonstrated superior performance over AMUPol for DNP-NMR in-cell studies.
• AMP-induced pore formation in bacteria correlated with increased signal enhancements and decreased T1 values for labeled Mac1.
• The DNP-NMR approach successfully provided enhanced signals and relaxation data from peptides within live bacterial cells.

Conclusions:

• This study establishes DNP-NMR with novel spin-labeled peptides as a viable method for in-cell structural studies of AMPs in bacteria.
• The findings pave the way for future in situ structural determination of AMPs interacting with their native bacterial targets.
• This technique holds significant potential for advancing our understanding of antibiotic mechanisms and developing new antimicrobial strategies.