title: Quantitative determination of lateral concentration and depth profile of histidine-tagged recombinant proteins probed by grazing incidence X-ray fluorescence.
abstract: We have demonstrated that the complementary combination of grazing incidence X-ray fluorescence (GIXF) with specular X-ray reflectivity (XRR) can be used to quantitatively determine the density profiles of Ni(2)(+) 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 Ni(2)(+) ions, yielding the thickness, roughness, and electron density of the stratified lipid monolayers. X-ray fluorescence intensities from Ni Kα 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 Ni(2)(+) ions associated with chelator headgroups with a high spatial accuracy (±5 Å). 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 Kα level were much weaker than Ni Kα signals, we could detect the location of S atoms in recombinant Xcad-11 proteins.
authors.affiliation: Physical Chemistry of Biosystems, Physical Chemistry Institute, University of Heidelberg, 69120 Heidelberg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Körner
authors.forename: Wasim
authors.initials: W
authors.lastname: Abuillan
authors.forename: Christina
authors.initials: C
authors.lastname: Deichmann
authors.forename: Fernanda F
authors.initials: FF
authors.lastname: Rossetti
authors.forename: Almut
authors.initials: A
authors.lastname: Köhler
authors.forename: Oleg V
authors.initials: OV
authors.lastname: Konovalov
authors.forename: Doris
authors.initials: D
authors.lastname: Wedlich
authors.forename: Motomu
authors.initials: M
authors.lastname: Tanaka
mesh_terms: D000388:Air; D000818:Animals; D015820:Cadherins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007477:Ions / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014867:Water / Q000737:chemistry; D014965:X-Rays; D014981:Xenopus; D029867:Xenopus Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D015820:Cadherins; C471213:His-His-His-His-His-His; D007477:Ions; D009842:Oligopeptides; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D014867:Water; C086960:osteoblast cadherin; D006639:Histidine; D009532:Nickel