Satsoura Dmitri, Kučerka Norbert, Shivakumar Sanjeevan, Pencer Jeremy, Griffiths Corrie, Leber Brian, Andrews David W, Katsaras John, Fradin Cécile
Department of Biochemistry and Biomedical Sciences, McMaster University Health Sciences Centre, Hamilton, Ontario, Canada.
Biochim Biophys Acta. 2012 Mar;1818(3):384-401. doi: 10.1016/j.bbamem.2011.10.007. Epub 2011 Oct 15.
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.
在凋亡刺激下，促凋亡蛋白Bax插入线粒体外膜，导致孔道形成并释放多种线粒体成分，从而决定细胞的命运。为了研究Bax与膜的结合，我们使用了一个体外系统，该系统由直径为50nm的脂质体组成，其脂质组成模拟线粒体膜，通过用纯化的tBid激活将重组纯化的全长Bax插入其中。我们使用荧光涨落方法检测蛋白质与膜的结合，发现它可以很好地用可溶性Bax和膜结合Bax之间的平衡来描述，并且在高蛋白与脂质体比例下，结合似乎在每50nm直径脂质体约15个Bax蛋白时达到饱和。然后，我们在不同的对比匹配条件下，使用小角中子散射获得了处于这种饱和结合状态的样品的结构数据。利用一个简单的模型来拟合中子数据，我们观察到与所有膜相关的Bax状态都是伞状的推测相反，大量蛋白质质量突出于膜上方。在蛋白质结合后，我们还观察到脂质双层变薄，同时脂质体半径增加，这种效应让人联想到抗菌肽对膜的作用。
