ESEEM Reveals Bound Substrate Histidine in the ABC Transporter HisQMP2

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 ( 2 H ESEEM) combined with site-directed spin labeling is a powerful tool to localize the substrate, histidine-d5, in the ABC transporter HisQMP 2 . Based on a homology model and spin label rotamer analyses, we calculated 2 H ESEEM spectra for eight possible labeling positions close to the putative substrate-binding site. Experimental 2 H ESEEM spectra were determined with spin labels bound either at position 169 of HisM, for which a detectable 2 H 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.