The proapoptotic protein tBid forms both superficially bound and membrane-inserted oligomers.
Bid is a proapopotic activator protein of the Bcl-2 family that plays a pivotal role in controlling mitochondrial outer membrane permeabilization during apoptosis. Here, we characterized the interaction of fluorescently labeled truncated Bid (tBid) with a mitochondria-like supported lipid bilayer at the single-molecule level. The proteins observed at the membrane exhibited a very wide range of mobility. Confocal images of the membrane displayed both diffraction-limited Gaussian spots and horizontal streaks, corresponding to immobile and mobile tBid species, respectively. We observed 1), fast-diffusing proteins corresponding to a loosely, probably electrostatically bound state; 2), slowly diffusing proteins, likely corresponding to a superficially inserted state; and 3), fully immobilized proteins, suggesting a fully inserted state. The stoichiometry of these proteins was determined by normalizing their fluorescence intensity by the brightness of a tBid monomer, measured separately using fluorescence fluctuation techniques. Strikingly, the immobile species were found to be mainly tetramers and higher, whereas the mobile species had on average a significantly lower stoichiometry. Taken together, these results show that as soluble Bid progresses toward a membrane-inserted state, it undergoes an oligomerization process similar to that observed for Bax.
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Sanjeevan
S
Shivakumar
Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, Canada.
Martin
M
Kurylowicz
Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, Canada.
Nehad
N
Hirmiz
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Yaseen
Y
Manan
Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, Canada.
Ouided
O
Friaa
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Aisha
A
Shamas-Din
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Pourya
P
Masoudian
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada; Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
Brian
B
Leber
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
David W
DW
Andrews
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada; Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, Canada. Electronic address: fradin@physics.mcmaster.ca.
Cécile
C
Fradin
D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D009068:Movement; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
D051023:BH3 Interacting Domain Death Agonist Protein; D008051:Lipid Bilayers