Nucleotide binding and self-stimulated GTPase activity of human guanylate-binding protein 1 (hGBP1).
The synthesis of human guanylate-binding protein 1 (hGBP1) is induced by interferon-gamma and its biological function is related to antiviral activity and regulation of proliferation. It interacts with guanine nucleotides, and its catalytic activity on GTP hydrolysis leads to the formation of phosphate ions and both GDP and GMP. Similar to other large GTPases like dynamin, hGBP1 shows higher specific GTP hydrolysis activity with increasing concentration of the protein. This is based on nucleotide-dependent self-association of hGBP1, which leads to self-stimulation of its GTPase activity. In this chapter we describe the characterization of the basic biochemical properties of hGBP1. Essentially, the biological activity of a GTPase is controlled by the type of nucleotide bound. Therefore, nucleotide binding is quantified in terms of affinity and dynamics since both of these aspects are important for the occurrence of hGBP1 bound to the one or the other nucleotide. In addition, we analyze the self-stimulated GTPase activity and show how to extract the hGBP1 homodimer dissociation constant from these data. Finally, with the help of size exclusion chromatography, nucleotide-dependent formation of hGBP1 dimers and tetramers is demonstrated. These biochemical characteristics may help to further understand the biological function of hGBP1.
Kunzelmann
Simone
S
Physikalische Chemie 1, Ruhr-Universität Bochum, Germany.
Praefcke
Gerrit J K
GJ
Herrmann
Christian
C
D002850:Chromatography, Gel; D005453:Fluorescence; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D062367:ortho-Aminobenzoates
C490287:GBP1 protein, human; D062367:ortho-Aminobenzoates; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins