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首页> 外文期刊>Biochemistry >Structural and Dynamic Perturbations Induced by Heme Binding in Cytochrome b_5
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Structural and Dynamic Perturbations Induced by Heme Binding in Cytochrome b_5

机译：血红素结合在细胞色素b_5中引起的结构和动态扰动。

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摘要

The water-soluble domain of rat hepatic cytochrome b5 undergoes marked structural changes upon heme removal. The solution structure of apocytochrome b5 shows that the protein is partially folded in the absence of the heme group, exhibiting a stable module and a disordered heme-binding loop. The quality of the apoprotein structure in solution was improved with the use of heteronuclear NMR data. Backbone amide hydrogen exchange was studied to characterize cooperative units in the protein. It was found that this criterion distinguished the folded module from the heme-binding loop in the apoprotein, in contrast to the holoprotein. The osmolyte trimethylamine N-oxide (TMAO) did not affect the structure of the apoprotein in the disordered region. TMAO imparted a small stabilization consistent with an unfolded state effect correlating with the extent of buried surface area in the folded region of the native apoprotein. The failure of the osmolyte to cause large conformational shifts in the disordered loop supported the view that the specificity of the local sequence for the holoprotein fold was best developed with the stabilization of the native state through heme binding. To dissect the role of the heme prosthetic group in forcing the disordered region into the holoprotein conformation, the axial histidine belonging to the flexible loop (His63) was replaced with an alanine, and the structural properties of the protein with carbon-monoxide-ligated reduced iron were studied. The His63Ala substitution resulted in a protein with lower heme affinity but nevertheless capable of complete refolding. This indicated that the coordination bond was not necessary to establish the structural features of the holoprotein. In addition, the weak binding of the heme in this protein resulted in conformational shifts at a location distant from the binding site. The data suggested an uneven distribution of cooperative elements in the structure of the cytochrome.
机译：大鼠肝细胞色素b5的水溶性域在去除血红素后会发生明显的结构变化。脱辅基细胞色素b5的溶液结构表明该蛋白质在不存在血红素基团的情况下被部分折叠，表现出稳定的模块和无序的血红素结合环。使用异核NMR数据可改善溶液中载脂蛋白结构的质量。研究了骨架酰胺氢交换以表征蛋白质中的协作单元。已发现与全蛋白相反，该标准将折叠的模块与载脂蛋白中的血红素结合环区分开。渗透液三甲胺N-氧化物（TMAO）不会影响无序区载脂蛋白的结构。 TMAO赋予了较小的稳定性，这与未折叠状态效应一致，该效应与天然载脂蛋白折叠区域中的隐埋表面积的大小有关。渗透压剂不能引起无序环中大构象变化的观点支持了这样的观点，即通过血红素结合使天然状态稳定可以最好地开发出针对全蛋白折叠的局部序列的特异性。为了剖析血红素修复基团在迫使无序区域进入全蛋白构象中的作用，将属于柔性环（His63）的轴向组氨酸替换为丙氨酸，并降低了与一氧化碳连接的蛋白质的结构特性研究了铁。 His63Ala取代产生的蛋白具有较低的血红素亲和力，但仍然能够完全重折叠。这表明配位键对于建立全蛋白的结构特征不是必需的。另外，血红素在该蛋白中的弱结合导致在远离结合位点的位置的构象转变。数据表明合作元素在细胞色素结构中分布不均。

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