title: Engineered fusion molecules at chelator lipid interfaces imaged by reflection interference contrast microscopy (RICM).
abstract: In molecular biology, biotechnology, and protein-engineering, the expression of histidine fusion proteins is a very powerful technique for the identification and one-step purification based on the interaction of the histidine stretch with immobilized metal complexes. By synthesis of a novel class of chelator lipids, this technique was combined with the concept of self-assembly leading to interfaces for immobilization and orientation of histidine-tagged biomolecules (Schmitt et al., 1994). Here, the chelator lipid layers were transferred onto solid substrate by vesicle fusion and Langmuir-Blodgett-techniques. Specific binding of a peptide containing an oligohistidine sequence to these functionalized interfaces was demonstrated by reflection interference contrast microscopy (RICM). Due to the phase separation behaviour of lipid mixtures, the chelator lipid interface could be further structured in two dimensions. Binding and organization of histidine-tagged molecules at these two-dimensional recognition arrays was imaged by RICM with a layer thickness resolution of 0.2 nm, and 0.5 microm laterally. Specific docking can be triggered by adding nickel ions and disrupted by EDTA. This concept opens up possibilities for reversible immobilization, enrichment and organization of histidine fusion proteins at interfaces and their application in biosensing.
authors.affiliation: Lehrstuhl für Biophysik E22, Technische Universitt München, Garching, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Gritsch
authors.forename: K
authors.initials: K
authors.lastname: Neumaier
authors.forename: L
authors.initials: L
authors.lastname: Schmitt
authors.forename: R
authors.initials: R
authors.lastname: Tampé
mesh_terms: D000595:Amino Acid Sequence; D002614:Chelating Agents; D006639:Histidine / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008857:Microscopy, Interference / Q000379:methods*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
chemical_list: D002614:Chelating Agents; D008051:Lipid Bilayers; D011993:Recombinant Fusion Proteins; D006639:Histidine