Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.
The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.
Department of Biochemistry, University of Toronto, Toronto, ON, Canada.|Department of Biological Sciences, Sunnybrook Research Institute, 2075 Bayview Ave., Room M7 621, Toronto, M4N 3M5, ON, Canada.
Justin P
JP
Pogmore
Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.|Department of Biological Sciences, Sunnybrook Research Institute, 2075 Bayview Ave., Room M7 621, Toronto, M4N 3M5, ON, Canada.
James M
JM
Pemberton
Department of Biological Sciences, Sunnybrook Research Institute, 2075 Bayview Ave., Room M7 621, Toronto, M4N 3M5, ON, Canada.|Department of Chemical Biology, McMaster University, Hamilton, ON, Canada.
Xiaoke
X
Chi
Department of Biochemistry, University of Toronto, Toronto, ON, Canada. david.andrews@sri.utoronto.ca.|Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada. david.andrews@sri.utoronto.ca.|Department of Biological Sciences, Sunnybrook Research Institute, 2075 Bayview Ave., Room M7 621, Toronto, M4N 3M5, ON, Canada. david.andrews@sri.utoronto.ca.
David W
DW
Andrews
D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D060066:Protein Interaction Maps; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism*
D051023:BH3 Interacting Domain Death Agonist Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D051020:bcl-X Protein; D045304:Cytochromes c
Apoptosis; Bcl-2 family; Detergent free; FRET; Förster-resonance energy transfer; Mitochondria; Protein interaction