Lecomte S, Hilleriteau C, Forgerit J P, Revault M, Baron M H, Hildebrandt P, Soulimane T
Laboratoire de Dynamique, Interactions et Réactivité, UMR-7075, CNRS-Université Paris VI, 2 rue Henri Dunant, 94320 Thiais, France.
Chembiochem. 2001 Mar 2;2(3):180-9. doi: 10.1002/1439-7633(20010302)2:3<180::aid-cbic180>3.3.co;2-2.
The structural changes of cytochrome c(552) bound to anionic and hydrophobic clay surfaces have been investigated by Fourier transform infrared spectroscopy. Binding to the anionic surface of montmorillonite is controlled by electrostatic interactions since addition of electrolyte (0.5 mol L(-1) KCl) causes desorption of more than 2/3 of the protein molecules. Electrostatic binding occurs through the back side of the protein (i.e., remote from the heme site) and is associated only with subtle changes of the secondary structure. In contrast, adsorption to the hydrophobic surface of talc leads to a decrease in alpha-helical structure by ca. 5% and an increase in beta-sheet structure by ca. 6%. These structural changes are attributed to a hydrophobic region on the front surface of cytochrome c(552) close to the partially exposed heme edge. This part on the protein surface is identified as the interaction domain for talc and most likely also serves for binding to the natural reaction partner, a ba(3)-oxidase. Fourier transform infrared spectra of cytochrome c(552) and the clay-cytochrome c(552) complexes have been measured as a function of time following dissolution and suspension in deuterated buffer, respectively. A two-dimensional correlation analysis was applied to these spectra to investigate the dynamics of the structural changes in the protein. For both complexes, adsorption and subsequent unfolding processes in the binding domains are faster than the time resolution of the spectroscopic experiments. Thus, the processes that could be monitored are refolding of peptide segments and side chain rearrangements following the adsorption-induced perturbation of the protein structure and the solvation of the adsorbed protein. In each case, side chain alterations of solvent-exposed tyrosine, aspartate, and glutamate residues were observed. For the cytochrome c(552)-talc complex, these changes are followed by a slow refolding of the peptide chain in the binding domain and, subsequently, a further H/D exchange of amide group protons.
通过傅里叶变换红外光谱研究了与阴离子和疏水粘土表面结合的细胞色素c(552)的结构变化。与蒙脱石阴离子表面的结合受静电相互作用控制，因为添加电解质（0.5 mol L(-1) KCl）会导致超过2/3的蛋白质分子解吸。静电结合通过蛋白质的背面（即远离血红素位点）发生，并且仅与二级结构的细微变化相关。相比之下，吸附到滑石的疏水表面会导致α-螺旋结构减少约5%，β-折叠结构增加约6%。这些结构变化归因于细胞色素c(552)前表面靠近部分暴露的血红素边缘的疏水区域。蛋白质表面的这一部分被确定为与滑石的相互作用域，很可能也用于与天然反应伙伴ba(3)-氧化酶结合。分别在氘代缓冲液中溶解和悬浮后，测量了细胞色素c(552)和粘土-细胞色素c(552)复合物的傅里叶变换红外光谱随时间的变化。对这些光谱进行二维相关分析，以研究蛋白质结构变化的动力学。对于这两种复合物，结合域中的吸附和随后的展开过程比光谱实验的时间分辨率快。因此，可以监测的过程是吸附诱导的蛋白质结构扰动后肽段的重新折叠和侧链重排以及吸附蛋白质的溶剂化。在每种情况下，都观察到了溶剂暴露的酪氨酸、天冬氨酸和谷氨酸残基的侧链变化。对于细胞色素c(552)-滑石复合物，这些变化之后是结合域中肽链的缓慢重新折叠，随后是酰胺基团质子的进一步H/D交换。
