ESEEM Reveals Bound Substrate Histidine in the ABC Transporter HisQMP2
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Localization of substrates in membrane proteins is an important but challenging task. In this paper, we show that deuterium electron spin echo envelope modulation spectroscopy (2H ESEEM) combined with site-directed spin labeling is a powerful tool to localize the substrate, histidine-d5, in the ABC transporter HisQMP2. Based on a homology model and spin label rotamer analyses, we calculated 2H ESEEM spectra for eight possible labeling positions close to the putative substrate-binding site. Experimental 2H ESEEM spectra were determined with spin labels bound either at position 169 of HisM, for which a detectable 2H ESEEM signal was calculated, or with a spin label bound at position 54 of HisQ as a negative control. The agreement between the calculated and experimental ESEEM spectra provides strong evidence for the histidine located in a binding site primarily liganded by residues of HisM as proposed by the homology model.
We thank Heidi Landmesser (Humboldt Universität zu Berlin) for excellent technical assistance. This work was supported by Alexander von Humboldt Foundation and DFG (STE640/10 to H.J.S. and SCHN274/16-1 to E.S.). Computational modeling was funded by the Russian Foundation for Basic Research grant 17-54-49004.
- 39.T. Eitinger, D.A. Rodionov, M. Grote, E. Schneider, F.E.M.S. Microbiol, Rev. 35, 3 (2011)Google Scholar