Clustering of Spin‐Labeled Molecules in Membranes: What Information Can Be Obtained From Electron–Electron Double Resonance

Clustering of antimicrobial peptides (AMPs) in bacterial membranes is a key step in the formation of nanoscale pores within them, which may underlie their bactericidal action. Double electron-electron resonance (DEER, also known as PELDOR) is a pulsed electron paramagnetic resonance (EPR) technique that has a unique ability to study distances between spin labels in the nanometer range. DEER has been successfully applied to the study of the conformations of doubly spin-labeled biomolecules; however, when applied to clusters with a number of spin labels N > 2, DEER encounters certain difficulties both in the proper implementation of measurements and in the interpretation of the data. This paper analyzes these emerging issues and provides recommendations on the proper use of the DEER method and the interpretation of its data when studying clusters of spin-labeled molecules. It demonstrates how the results of studies of AMP clusters in model membranes can be reinterpreted.