In-situ spin labeling of his-tagged proteins: distance measurements under in-cell conditions.

New spin labeling strategies have immense potential in studying protein structure and dynamics under physiological conditions with electron paramagnetic resonance (EPR) spectroscopy. Here, a new spin-labeled chemical recognition unit for switchable and concomitantly high affinity binding to His-tagged proteins was synthesized. In combination with an orthogonal site-directed spin label, this novel spin probe, Proxyl-trisNTA (P-trisNTA) allows the extraction of structural constraints within proteins and macromolecular complexes by EPR. By using the multisubunit maltose import system of E.coli: 1)the topology of the substrate-binding protein, 2)its substrate-dependent conformational change, and 3)the formation of the membrane multiprotein complex can be extracted. Notably, the same distance information was retrieved both in vitro and in situ allowing for site-specific spin labeling in cell lysates under in-cell conditions. This approach will open new avenues towards in-cell EPR.