The biochemical and structural characterization of membrane proteins requires extraction from their native membranes with detergents, often followed by reconstitution into liposomes or nanodiscs to mimic the lipid bilayer. However, to fully understand the biophysical properties of membrane proteins, they should be ideally investigated within living cells. Site-directed spin labeling Electron Paramagnetic Resonance (EPR) spectroscopy is one of the techniques developing tools for in cell applications. Yet, it is limited in terms of sensitivity and fidelity when it comes to membrane proteins in vivo due to the lack of methods to introduce spectroscopic labels at specific positions inside the cell. Here, we generated a spin-labeled nanobody as structural probe to interrogate the conformational cycle of the ABC transporter MsbA in vitro and in cell by Double Electron Electron Resonance (DEER). Surprisingly, in metabolically active cells we identified a wide inward-open conformation of MsbA, which is commonly considered a non-physiological state. A comparative DEER and cryo-EM analysis shed light on the funneling towards a narrow inward-open conformation of MsbA induced by nanodisc reconstitution. This study paves the way towards the use of spin-labeled nanobodies for in-cell EPR applications on membrane proteins^1,2.

[1]   L. Galazzo, G. Meier, M.H. Timachi, C.A.J. Hutter, M.A. Seeger, E. Bordignon PNAS, 2020, 117(5):2441-2448.
[2]   L. Galazzo, G. Meier et al. in preparation