title: Nonequivalence of the nucleotide-binding subunits of an ABC transporter, the histidine permease, and conformational changes in the membrane complex.
abstract: The membrane-bound complex of the Salmonella typhimurium histidine permease, an ABC transporter (or traffic ATPase), is composed of two membrane proteins, HisQ and HisM, and two identical copies of an ATP-hydrolyzing protein, HisP. We have developed a technique that monitors quantitatively the sulfhydryl modification levels within the intact complex, and we have used it to investigate whether the HisP subunits behave identically within the complex. We show here that they interact differently with various thiol-specific reagents, thus indicating that, despite being identical, they are arranged asymmetrically. The possible basis of this asymmetry is discussed. We have also analyzed the occurrence of conformational changes during various stages of the activity cycle using thiol-specific reagents, fluorescence measurements, and circular dichroism spectroscopy. Cys-51, located close to the ATP-binding pocket, reflects conformational changes upon binding of ATP but does not participate in changes involved in signaling and translocation. The latter are shown to cause secondary structure alterations, as indicated by changes in alpha-helices; tertiary structure alterations also occur, as shown by fluorescence studies.
authors.affiliation: Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720, USA.
authors.forename: D I
authors.initials: DI
authors.lastname: Kreimer
authors.forename: K P
authors.initials: KP
authors.lastname: Chai
authors.forename: G
authors.initials: G
authors.lastname: Ferro-Luzzi Ames
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D026923:Amino Acid Transport Systems, Basic; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D001426:Bacterial Proteins; D001643:Bridged Bicyclo Compounds / Q000378:metabolism; D002942:Circular Dichroism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D011487:Protein Conformation / Q000528:radiation effects; D017433:Protein Structure, Secondary / Q000528:radiation effects; D012486:Salmonella typhimurium / Q000201:enzymology / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D001643:Bridged Bicyclo Compounds; D046911:Macromolecular Substances; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; C043828:histidine permease, Bacteria; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C028381:monobromobimane