title: In-situ spin labeling of his-tagged proteins: distance measurements under in-cell conditions.
abstract: 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.
authors.affiliation: Institute of Biochemistry, Biocenter, Cluster of Excellence-Macromolecular Complexes (CEF-MC), Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt a.M. (Germany).
authors.forename: Christoph
authors.initials: C
authors.lastname: Baldauf
authors.forename: Katrin
authors.initials: K
authors.lastname: Schulze
authors.forename: Petra
authors.initials: P
authors.lastname: Lueders
authors.forename: Enrica
authors.initials: E
authors.lastname: Bordignon
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D008320:Maltose / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013113:Spin Labels
chemical_list: D011506:Proteins; D013113:Spin Labels; D008320:Maltose
keywords: chelators; electron paramagnetic resonance; membrane proteins; molecular recognition; spin probes