Coexistence of Native-Like and Non-Native Cytochrome c on Anionic Liposomes with Different Cardiolipin Content
Coexistence of Native-Like and Non-Native Cytochrome c on Anionic Liposomes with Different Cardiolipin Content
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DOI：
10.1021/acs.jpcb.5b07328
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发表时间：
2015-10-08
影响因子：
3.3
通讯作者：
Schweitzer-Stenner, Reinhard
中科院分区：
文献类型：
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作者：
Pandiscia, Leah A.;Schweitzer-Stenner, Reinhard
我们采用荧光，可见圆二色谱和吸收光谱的组合，研究铁细胞色素c的构象变化后，其结合到含心磷脂的小单层囊泡。作为心磷脂浓度、脂质体的心磷脂含量和溶剂的NaCl浓度的函数进行测量。用一种新的模型分析了数据，该模型将单个结合步骤与结合到膜表面的天然样和非天然样蛋白质之间的构象平衡相结合。这两种构象之间的平衡，这本身是由结构上略有不同的亚构象，转移到更非天然样的构象增加心磷脂浓度。对于本文中所描述的结合等温线，我们明确地考虑了在低脂质浓度和脂质体表面的高占有率下分子拥挤对蛋白质结合的熵贡献。增加脂质体的CL含量增加了总体结合亲和力，但使构象分布更容易受到钠离子和氯离子的影响，这使平衡向更天然的状态移动，并直接抑制结合，特别是与100%心磷脂含量的脂质体的结合。光谱证据进一步表明，一小部分的非本地构象采用pentacocordinated状态类似于C类过氧化物酶中获得的。我们的研究结果的基础上，我们提出了一个假设，描述了促进和阻碍力之间的平衡控制细胞色素c的过氧化物酶活性的内膜空间的线粒体。
We employed a combination of fluorescence, visible circular dichroism, and absorption spectroscopy to study the conformational changes of ferricytochrome c upon its binding to cardiolipin-containing small unilamellar vesicles. The measurements were performed as a function of the cardiolipin concentration, the cardiolipin content of the liposomes, and the NaCl concentration of the solvent. The data were analyzed with a novel model that combines a single binding step with a conformational equilibrium between native-like and non-native-like proteins bound to the membrane surface. The equilibrium between the two conformations, which themselves are comprised of structurally slightly different subconformations, shifts to the more non-native-like conformation with increasing cardiolipin concentration. For the binding isotherms described in this paper, we explicitly considered the enthalpic and entropic contributions of molecular crowding to protein binding at low lipid concentrations and high occupancy of the liposome surface. Increasing the CL content of liposomes increases the overall binding affinity but makes the conformational distribution much more susceptible to the influence of sodium and chloride ions, which shifts the equilibrium toward the more native-like state and directly inhibits binding, particularly to liposomes with 100% cardiolipin content. Spectroscopic evidence further suggests that a fraction of the non-native conformers adopts a pentacoordinated state similar to those obtained in class C peroxidases. On the basis of our results, we propose a hypothesis that describes the balance between facilitating and impeding forces controlling the peroxidase activity of cytochrome c in the inner membrane space of mitochondria.