title: Functional immobilization of a DNA-binding protein at a membrane interface via histidine tag and synthetic chelator lipids.
abstract: The coupling of a DNA-binding protein to self-organized lipid monolayers is examined at the air-water interface by means of film balance techniques and epifluorescence microscopy. We used two recombinant species of the heat shock factor HSF24 which differ only in a carboxy-terminal histidine tag that interacts specifically with the nickel-chelating head group of a synthetic chelator lipid. As key function, HSF24 binds to DNA that contains heat-shock responsible promoter elements. In solution, DNA-protein complex formation is demonstrated for the wild type and fusion protein. Substantial questions of these studies are whether protein function is affected after adsorption to lipid layers and whether a specific docking via histidine tag to the chelator lipid leads to functional immobilization. Using lipid mixtures that allow a lateral organization of chelator lipids within the lipid film, specific binding and unspecific adsorption can be distinguished by pattern formation of DNA-protein complexes. At the lipid interface, functional DNA-protein complexes are only detected, when the histidine-tagged protein was immobilized specifically to a chelator lipid containing monolayer. These results demonstrate that the immobilization of histidine-tagged biomolecules to membranes via chelator lipids is a promising approach to achieve a highly defined deposition of these molecules at an interface maintaining their function.
authors.affiliation: Physik Department, Technische Universität München, Garching, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Dietrich
authors.forename: O
authors.initials: O
authors.lastname: Boscheinen
authors.forename: K D
authors.initials: KD
authors.lastname: Scharf
authors.forename: L
authors.initials: L
authors.lastname: Schmitt
authors.forename: R
authors.initials: R
authors.lastname: Tampé
mesh_terms: D000327:Adsorption; D000588:Amines; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002614:Chelating Agents / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000493:pharmacokinetics; D006639:Histidine; D050356:Lipid Metabolism; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D010455:Peptides / Q000378:metabolism; D010940:Plant Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000493:pharmacokinetics
chemical_list: D000588:Amines; D002614:Chelating Agents; C098034:HSF24 protein, Lycopersicon peruvianum; D006360:Heat-Shock Proteins; D008567:Membranes, Artificial; D010455:Peptides; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C095818:nitrilotriacetic acid dioctadecylamine; C033223:polyhistidine; D006639:Histidine; D009571:Nitrilotriacetic Acid