In-situ observation of conformational dynamics and protein-ligand/substrate interactions in outer membrane proteins with DEER/PELDOR spectroscopy

Observing structure and conformational dynamics of membrane proteins at high-resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer membrane proteins (OMPs) in intact E. coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in-situ labeling of engineered cysteines with a methanethiosulfonate functionalized nitroxide spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OM) under selective conditions. The interspin distances are measured in-situ using pulsed electron-electron double resonance spectroscopy (PELDOR or DEER). The residual background signals, which are problematic for in-situ structural biology, contributes specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5–7 d and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and the data analysis typically take 4–5 d. The described protocol provides a general strategy for observing protein-ligand/substrate interactions, oligomerization, and conformational dynamics of OMPs in the native OM and intact E. coli.