tructure and dynamics of the immuno-active GTPase hGBP1 studied by single molecule FRET and DEER EPR

Upon penetration of the human body with infectious pathogens a distinct upregulation of Interferon gamma, an inflammatory cytokine, leads to a high level expression of the human guanylate binding protein 1 (hGBP1) in the target cell. Cell biological studies revealed a broad diversity of defensive effects (antiviral and -bacterial) as well as hGBP1 being a potential tumor marker [1,2,3]. In vitro oligomers are formed upon GTP binding and hydrolysis in a highly protein concentration dependent manner which in return increases the nucleotide hydrolysis rate [3]. It was found that this process involves also extensive conformational changes within the protein [4]. To study the intra- and intermolecular conformational changes in the presence of different nucleotide analogues, ensemble and multiparameter fluorescence detection under single molecule conditions as well as lifetime measurements and DEER-EPR were applied. For maleimide labeling cysteines were introduced at distinct positions by site-directed mutagenesis and coupled to either a FRET dye pair or EPR spin label. It could be shown that the nucleotide-free state of hGBP1 fluctuates around the conformation seen in x-ray data. In the presence of the nucleotide analogues GppNHp/GTPγS or GDP AlFx conformational changes especially concerning the two C-terminal helices 12 and 13 were observed. Various intramolecular and intermolecular distances could be extracted. These data allow conclusions about the overall oligomerisation-dependent structural and dynamic changes inside hGBP1.