title: A homogeneous fluorescence resonance energy transfer system for monitoring the activation of a protein switch in real time.
abstract: A homogeneous fluorescence resonance energy transfer (FRET) system for the real-time monitoring of exchange factor-catalyzed activation of a ras-like small GTPase is described. The underlying design is based on supramolecular template effects exerted by protein-protein interactions between the GTPase adenosine diphosphate ribosylation factor (ARF) and its effector protein GGA3. The GTPase is activated when bound to guanosine triphosphate (GTP) and switched off in its guanosine diphosphate (GDP)-bound state. Both states are accompanied by severe conformational changes that are recognized by GGA3, which only binds the GTPase "on" state. GDP-to-GTP exchange, i.e., GTPase activation, is catalyzed by the guanine nucleotide exchange factor cytohesin-2. When GGA3 and the GTPase ARF1 are labeled with thoroughly selected FRET probes, with simultaneous recording of the fluorescence of an internal tryptophan residue in ARF1, the conformational changes during the activation of the GTPase can be monitored in real time. We applied the FRET system to a multiplex format that allows the simultaneous identification and distinction of small-molecule inhibitors that interfere with the cytohesin-catalyzed ARF1 activation and/or with the interaction between activated ARF1-GTP and GGA3. By screening a library of potential cytohesin inhibitors, predicted by in silico modeling, we identified new inhibitors for the cytohesin-catalyzed GDP/GTP exchange on ARF1 and verified their increased potency in a cell proliferation assay.
authors.affiliation: LIMES Institute, Chemical Biology and Medicinal Chemistry Unit, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Bill
authors.forename: Heike
authors.initials: H
authors.lastname: Blockus
authors.forename: Dagmar
authors.initials: D
authors.lastname: Stumpfe
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bajorath
authors.forename: Anton
authors.initials: A
authors.lastname: Schmitz
authors.forename: Michael
authors.initials: M
authors.lastname: Famulok
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000737:chemistry* / Q000494:pharmacology; D033942:Adaptor Proteins, Vesicular Transport / Q000737:chemistry* / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D020690:GTPase-Activating Proteins / Q000737:chemistry; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D014364:Tryptophan / Q000737:chemistry
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C102474:cytohesin-2; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D020558:GTP Phosphohydrolases; D020823:ADP-Ribosylation Factor 1