using flim-fret to measure conformational changes of transglutaminase type 2 in live cells使用flim-fret测量2型在活细胞转谷氨酰胺酶的构象变化.pdfVIP

Using FLIM-FRET to Measure Conformational Changes of Transglutaminase Type 2 in Live Cells 1 1 2 1 Nicholas S. Caron , Lise N. Munsie , Jeffrey W. Keillor , Ray Truant * 1 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada, 2 Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada Abstract Transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that ¨ allows for quantitative assessment of TG2 conformational changes in live cells using Forster resonance energy transfer (FRET), as measured by fluorescence lifetime imaging microscopy (FLIM). Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible