Human guanylate-binding protein 1 as a model system investigated by several surface techniques

In medical technologies concerning the surface immobilization of proteins in a defined orientation, maintaining their activity is a critical aspect. Therefore, in this study, the authors have investigated the activity of an elongated protein attached to a self-assembled monolayer supported streptavidin layer for different relative orientations of the protein with regard to the surface. Several mutants of this protein, human guanylate-binding protein 1 (hGBP1) showing GTPase catalytic activity, have been furnished with either one or two biotin anchors. Various independent methods that are based on different biophysical properties such as surface plasmon resonance, atomic force microscopy, and quartz crystal microbalance have been used to determine the orientation of the hGBP1 variants after anchoring them via a streptavidin-linker to a biotinylated surface. The activity of guanosine-triphosphate hydrolysis of hGBP1 monomers bound on the surface is found to depend on their orientation relative to the substrate, relating to their ability to form dimers with other neighboring anchored mutants; the maximum activity is lower than that observed in solutions, as might be expected from diffusion limitations at the solid/liquid interface on the one hand and prevention from homodimer formation due to immobilization on the other hand.