title: Fabrication of Nanometer- and Micrometer-Scale Protein Structures by Site-Specific Immobilization of Histidine-Tagged Proteins to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting Groups.
abstract: The site-specific immobilization of histidine-tagged proteins to patterns formed by far-field and near-field exposure of films of aminosilanes with protein-resistant photolabile protecting groups is demonstrated. After deprotection of the aminosilane, either through a mask or using a scanning near-field optical microscope, the amine terminal groups are derivatized first with glutaraldehyde and then with N-(5-amino-1-carboxypentyl)iminodiacetic acid to yield a nitrilo-triacetic-acid-terminated surface. After complexation with Ni(2+), this surface binds histidine-tagged GFP and CpcA-PEB in a site-specific fashion. The chemistry is simple and reliable and leads to extensive surface functionalization. Bright fluorescence is observed in fluorescence microscopy images of micrometer- and nanometer-scale patterns. X-ray photoelectron spectroscopy is used to study quantitatively the efficiency of photodeprotection and the reactivity of the modified surfaces. The efficiency of the protein binding process is investigated quantitatively by ellipsometry and by fluorescence microscopy. We find that regions of the surface not exposed to UV light bind negligible amounts of His-tagged proteins, indicating that the oligo(ethylene glycol) adduct on the nitrophenyl protecting group confers excellent protein resistance; in contrast, exposed regions bind His-GFP very effectively, yielding strong fluorescence that is almost completely removed on treatment of the surface with imidazole, confirming a degree of site-specific binding in excess of 90%. This simple strategy offers a versatile generic route to the spatially selective site-specific immobilization of proteins at surfaces.
authors.affiliation: Department of Chemistry, University of Sheffield , Sheffield S3 7HF, United Kingdom.
authors.forename: Sijing
authors.initials: S
authors.lastname: Xia
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield , Western Bank, Sheffield S10 2TN, United Kingdom.
authors.forename: Michaël
authors.initials: M
authors.lastname: Cartron
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield , Western Bank, Sheffield S10 2TN, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Morby
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University , University Park, Pennsylvania 16802, United States.|Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Donald A
authors.initials: DA
authors.lastname: Bryant
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield , Western Bank, Sheffield S10 2TN, United Kingdom.
authors.forename: C Neil
authors.initials: CN
authors.lastname: Hunter
authors.affiliation: Department of Chemistry, University of Sheffield , Sheffield S3 7HF, United Kingdom.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Leggett
mesh_terms: D001665:Binding Sites; D006639:Histidine / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry*; D055616:Microtechnology; D049329:Nanostructures / Q000737:chemistry*; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D055668:Photochemical Processes; D012833:Siloxanes / Q000737:chemistry*
chemical_list: D055767:Immobilized Proteins; D012833:Siloxanes; D006639:Histidine; D009571:Nitrilotriacetic Acid
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