Interaction of the full-length Bax protein with biomimetic mitochondrial liposomes: a small-angle neutron scattering and fluorescence study.
In response to apoptotic stimuli, the pro-apoptotic protein Bax inserts in the outer mitochondrial membrane, resulting in the formation of pores and the release of several mitochondrial components, and sealing the cell's fate. To study the binding of Bax to membranes, we used an in vitro system consisting of 50nm diameter liposomes prepared with a lipid composition mimicking that of mitochondrial membranes in which recombinant purified full-length Bax was inserted via activation with purified tBid. We detected the association of the protein with the membrane using fluorescence fluctuation methods, and found that it could well be described by an equilibrium between soluble and membrane-bound Bax and that at a high protein-to-liposome ratio the binding seemed to saturate at about 15 Bax proteins per 50nm diameter liposome. We then obtained structural data for samples in this saturated binding regime using small-angle neutron scattering under different contrast matching conditions. Utilizing a simple model to fit the neutron data, we observed that a significant amount of the protein mass protrudes above the membrane, in contrast to the conjecture that all of the membrane-associated Bax states are umbrella-like. Upon protein binding, we also observed a thinning of the lipid bilayer accompanied by an increase in liposome radius, an effect reminiscent of the action of antimicrobial peptides on membranes.
Department of Biochemistry and Biomedical Sciences, McMaster University Health Sciences Centre, Hamilton, Ontario, Canada.
Dmitri
D
Satsoura
Norbert
N
Kučerka
Sanjeevan
S
Shivakumar
Jeremy
J
Pencer
Corrie
C
Griffiths
Brian
B
Leber
David W
DW
Andrews
John
J
Katsaras
Cécile
C
Fradin
D006801:Humans; D008081:Liposomes / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry*; D008958:Models, Molecular; D033363:Neutron Diffraction / Q000379:methods; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D051028:bcl-2-Associated X Protein / Q000737:chemistry*
C494826:BAX protein, human; D008081:Liposomes; D051028:bcl-2-Associated X Protein