Quantitative Determination of Lateral Concentration and Depth Profile of Histidine-Tagged Recombinant Proteins Probed by Grazing Incidence X-ray Fluorescence

We have demonstrated that the complementary combination of grazing incidence X-ray fluorescence (GIXF) with specular X-ray reflectivity (XMR) can be used to quantitatively determine the density profiles of Ni2+ ions complexed with chelator headgroups as well as S atoms in recombinant proteins anchored to lipid monolayers at the air/water interface. First, we prepared phospholipid monolayers incorporating. chelator lipid anchors at different molar fractions at the air/water interface. The fine structures perpendicular to the global plane of monolayers were characterized by XRR in the presence of Ni2+ ions, yielding the thickness, roughness, and electron density of the stratified lipid monolayers. X-ray fluorescence intensities from Ni K alpha core levels recorded at the incidence angles below and above the critical angle of total reflection allow for the determination of the position and lateral density of Ni2+ ions associated with chelator headgroups with a high spatial accuracy (+/- 5 angstrom). The coupling of histidine-tagged Xenopus cadherin 11 (Xcad-11) can also be identified by changes in the fines structures using XRR. Although fluorescence intensities from S K alpha level were much weaker than Ni K alpha signals, we could detect the location of S atoms in recombinant Xcad-11 proteins.