title: Immobilization of biotinylated hGBP1 in a defined orientation on surfaces is crucial for uniform interaction with analyte proteins and catalytic activity.
abstract: Guanylate binding proteins (GBPs) belong to the dynamin superfamily of large GTP binding proteins. A biochemical feature common to these proteins is guanosine-triphosphate (GTP) binding leading to self-assembly of the proteins, and this in turn results in higher catalytic GTP hydrolysis activity. In the case of human guanylate binding protein 1 (hGBP1) homodimer formation is observed after binding of nonhydrolyzable GTP analogs like GppNHp. hGBP1 is one of seven GBP isoforms identified in human. While cellular studies suggest heterocomplex formation of various isoforms biochemical binding studies in quantitative terms are lacking. In this work we established a method to study hGBP1 interactions by attaching this protein in a defined orientation to a surface allowing for interaction with molecules from the solution. Briefly, specifically biotinylated hGBP1 is attached to a streptavidin layer on a self-assembled monolayer (SAM) surface allowing for characterization of the packing density of the immobilized protein by surface plasmon resonance (SPR) technology and atomic force microscopy (AFM), respectively. In addition, the enzymatic activity of immobilized hGBP1 and the kinetics of interaction with binding partners in solution are quantified. We present a procedure for attaching an enzyme in a defined orientation to a surface which exposes its active end, the GTPase domain to the solution resulting in a homogeneous population of this enzyme in terms of enzymatic activity and of interaction with soluble proteins.
authors.affiliation: Department of Physical Chemistry I, University of Bochum, 44801 Bochum, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Syguda
authors.forename: Andreas
authors.initials: A
authors.lastname: Kerstan
authors.forename: Tatjana
authors.initials: T
authors.lastname: Ladnorg
authors.forename: Florian
authors.initials: F
authors.lastname: Stüben
authors.forename: Christof
authors.initials: C
authors.lastname: Wöll
authors.forename: Christian
authors.initials: C
authors.lastname: Herrmann
mesh_terms: D055162:Biocatalysis; D019921:Biotinylation; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; C490287:GBP1 protein, human; D019809:Streptavidin; D019204:GTP-Binding Proteins