Immobilization of Biotinylated hGBP1 in a Defined Orientation on Surfaces Is Crucial for Uniform Interaction with Analyte Proteins and Catalytic Activity

摘要

Guanylate binding proteins (GBPs) belong to the dynaminnsuperfamily of large GTP binding proteins. A biochemical feature commonnto these proteins is guanosine-triphosphate (GTP) binding leading to selfassemblynof the proteins, and this in turn results in higher catalytic GTPnhydrolysis activity. In the case of human guanylate binding protein 1n(hGBP1) homodimer formation is observed after binding of nonhydrolyzablenGTP analogs like GppNHp. hGBP1 is one of seven GBPnisoforms identified in human. While cellular studies suggest heterocomplexnformation of various isoforms biochemical binding studies in quantitativenterms are lacking. In this work we established a method to study hGBP1ninteractions by attaching this protein in a defined orientation to a surfacenallowing for interaction with molecules from the solution. Briefly,nspecifically biotinylated hGBP1 is attached to a streptavidin layer on a self-assembled monolayer (SAM) surface allowing forncharacterization of the packing density of the immobilized protein by surface plasmon resonance (SPR) technology and atomicnforce microscopy (AFM), respectively. In addition, the enzymatic activity of immobilized hGBP1 and the kinetics of interactionnwith binding partners in solution are quantified. We present a procedure for attaching an enzyme in a defined orientation to ansurface which exposes its active end, the GTPase domain to the solution resulting in a homogeneous population of this enzymenin terms of enzymatic activity and of interaction with soluble proteins.