pH-Induced Switch between Different Modes of Cytochrome c Binding to Cardiolipin-Containing Liposomes
pH-Induced Switch between Different Modes of Cytochrome c Binding to Cardiolipin-Containing Liposomes
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DOI：
10.1021/acsomega.8b02574
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发表时间：
2019-01-01
期刊：
影响因子：
4.1
通讯作者：
Malyshka, Dmitry
中科院分区：
文献类型：
--
作者：
Milorey, Bridget;Schweitzer-Stenner, Reinhard;Malyshka, Dmitry
结合荧光、可见圆二色性（CD）、吸收和共振拉曼光谱技术，研究了铁细胞色素c与含心磷脂脂质体（20%1，1 '，1，2'-四油酰心磷脂和1，2-去油酰-sn-甘油-3-磷酸胆碱）在酸性pH（6.5）下结合后的结构变化。根据Kawai等人的早期工作[J.Biol.Chem.2005，280，34709-347171]，在该pH下的细胞色素c结合受心磷脂的磷酸头基和所谓L-位点的氨基酸侧链之间的相互作用控制，所述L-位点含有带电残基K22、K25、K27，以及潜在的H26和H33。我们发现，L-位点结合导致构象转变，涉及蛋白质的连接和自旋状态的变化。在本文中，我们报告的光谱响应越来越多的含心磷脂的脂质体在pH值为6.5的情况下和存在下的NaCl。发现后者在50 mM浓度下已经主要抑制蛋白结合。抑制效果可以通过应用Heimburg等人的静电理论定量再现[Biophys. J. 1995，68，536-546]。与相应的光谱响应数据在pH 7.4下获得的比较显示，后者表明疏水结合，其次是静电驱动的构象变化的主要差异。可见的CD数据表明，脂质体结合的铁细胞色素C的血红素口袋中的结构变化在一定程度上类似于那些在中性和酸性pH值的尿素中的变性蛋白质。测量的吸收和CD Soret带的细胞色素C在一个大的访问的存在下，在膜表面的电场引起的不一致。它对血红素口袋中的内部电场的贡献可以通过光谱手段检测到，这一事实表明蛋白质渗透到膜表面。
Fluorescence, visible circular dichroism (CD), absorption, and resonance Raman spectroscopy techniques were combined to explore structural changes of ferricytochrome c upon its binding to cardiolipin-containing liposomes (20% 1,1', 1,2'-tetraoleyolcardiolipin and 1,2-deoleyol-sn-glycero-3-phosphocholine) at acidic pH (6.5). According to the earlier work of Kawai et al. [J. Biol. Chem. 2005, 280, 34709-347171], cytochrome c binding at this pH is governed by interactions between the phosphate head groups of cardiolipin and amino acid side chains of the so-called L-site, which contains the charged residues K22, K25, K27, and potentially H26 and H33. We found that L-site binding causes a conformational transition that involves a change of the protein's ligation and spin state. In this paper, we report spectroscopic responses to an increasing number of cardiolipin-containing liposomes at pH 6.5 in the absence and presence of NaCl. The latter was found to mostly inhibit protein binding already with 50 mM concentration. The inhibition effect can be quantitatively reproduced by applying the electrostatic theory of Heimburg et al. [Biophys. J. 1995, 68, 536-546]. A comparison with corresponding spectroscopic response data obtained at pH 7.4 reveals major differences in that the latter indicates hydrophobic binding, followed by an electrostatically driven conformational change. Visible CD data suggest that structural changes in the heme pocket of liposome-bound ferricytochrome c resemble to some extent those in the denatured protein in urea at neutral and acidic pH. The measured noncoincidence between absorption and CD Soret band of cytochrome c in the presence of a large access of cardiolipin is caused by the electric field at the membrane surface. The very fact that its contribution to the internal electric field in the heme pocket is detectable by spectroscopic means suggests some penetration of the protein into membrane surface.