Falzone C J, Mayer M R, Whiteman E L, Moore C D, Lecomte J T
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1996 May 28;35(21):6519-26. doi: 10.1021/bi960501q.
In order to characterize the structural and dynamic factors that determine the assembly in b hemoproteins, the solution structure of the 98-residue protein apocytochrome b5 was determined by NMR methods. Over 800 experimental restraints derived from a series of two- and three-dimensional experiments were used. Holocytochrome b5, the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequence the following elements of secondary structure: beta 1-alpha 1-beta 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Mathews, F.S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp. 107-147, Academic Press, New York]. The folded holoprotein possesses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (core 2). The apoprotein was found to contain a stable four-stranded beta-sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha-helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short alpha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme binding pocket and are disordered in the apoprotein NMR structure. According to backbone 1H-15N NOE results, the most flexible region of the apoprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located immediately prior to alpha 5) exhibits faster internal motions than that bearing His-39 (at the C-terminal end of alpha 2). The latter imidazole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded that heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guidelines for the rational design of b hemoproteins: a modular structure including a packed, stable core and a partially folded binding site is anticipated to present strong kinetic and thermodynamic advantages compared to approaches relying on the complete formation of secondary structure prior to heme binding.
为了表征决定b族血红蛋白组装的结构和动力学因素，采用核磁共振方法测定了98个残基的脱辅基细胞色素b5蛋白的溶液结构。使用了来自一系列二维和三维实验的800多个实验约束条件。全细胞色素b5蛋白中，铁原卟啉-IX与His-39和His-63配位，其二级结构序列包含以下元件：β1-α1-β4-β3-α2-α3-β5-α4-α5-β2-α6[马修斯，F.S.，切尔文斯基，E.W.，&阿戈斯，P.（1979年）《卟啉》，第7卷，第107 - 147页，学术出版社，纽约]。折叠后的全蛋白具有两个疏水核心：围绕血红素的一个广泛的功能性核心（核心1），以及远离血红素的一个较小的结构核心（核心2）。发现脱辅基蛋白包含一个稳定的四链β折叠片层，包括β1、β2、β3和β4以及三个α螺旋，分别对应α1、α2和α6。两个短α螺旋（α3和α5）似乎部分形成，未检测到α4。这三个螺旋和β5围绕血红素结合口袋，在脱辅基蛋白的核磁共振结构中无序。根据主链1H-15N NOE结果，脱辅基蛋白除末端外最灵活的区域从Ala-50（在β5中）延伸至Glu-69（在α5中）。携带His-63的多肽片段（位于α5之前）的内部运动比携带His-39的片段（在α2的C末端）更快。后者的咪唑占据一个受限的空间区域，而前者相对于稳定核心可以采取多种取向。得出的结论是，血红素去除影响了核心1的大部分结构和动力学，而核心2基本保持完整。这些结果为b族血红蛋白的合理设计提供了指导：与依赖于血红素结合前二级结构完全形成的方法相比，一种包括紧密堆积的稳定核心和部分折叠的结合位点的模块化结构预计具有强大的动力学和热力学优势。
