Invited Speaker 23rd International Society of Magnetic Resonance Conference 2023

The use of dynamic nuclear polarization (DNP) NMR to investigate biologically active molecules in native cell environments is rapidly offering unprecedented levels of structural and functional details. Efforts to characterise the radical lifetime and its cellular location, the cell integrity and survival rate, together with engineering of in-cell protein production have generated multiple methods to perform in-cell DNP NMR. Thus, we previously have shown the benefit of using spin labelled peptides for targeted DNP NMR in E. coli systems. The protocol led to localized enhancement of the NMR signals at the membrane interface, and improved prospects for understanding the mechanism of action of membrane-active peptides, such as antimicrobial peptides (AMPs). While information at the membrane interface is of particular interest for AMP studies, other internal structures have been shown to be perturbed and to contribute to the death of bacteria. Thus, a more global monitoring of the impact of AMPs on bacteria could provide new insights in their mode of action. We will show that untargeted DNP NMR is a useful tool to globally monitor the effects of AMPs on bacteria by using ¹⁵N labelled E. coli (Fig. 1). The non-specific ¹⁵N labelling allows simultaneous observation of nucleic acids, proteins and lipids in-cell. We will show how several AMPs have different impacts on these structures. For instance, some AMPs disrupted the lipid packing while some also perturbed the nucleic acids. Overall, the ability to monitor the action of antimicrobial peptides in situ provides greater insight into their multi-impact mode of action.

Figure 1 DNP-enhanced ¹⁵N CPMAS spectra of untreated E. coli cells (black line) and in the presence of Mac1 at 15:1 w/w ratio (red line). The left panel is scaled 4-fold compared to the right panel to increase visibility. The DNA bases, amino acids with nitrogen containing sidechains and the phospholipid palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE) structures are displayed in the inserts with nitrogen (blue), oxygen (red), phosphorous (orange), carbon (grey) and hydrogen (white) atoms.