STRUCTURE/FUNCTION OF MEMBRANE BOUND CYTOCHROMES

高度保守的细胞色素b6f复合体和反应中心 氧合作用的细胞色素b-559将用于基础研究 整膜细胞色素的结构和功能方面。 建议的研究基于前一次拨款的以下结果 支持：(I)在2.3A分辨率下的晶体结构的溶液 细胞色素f的活性主要外在结构域，第一结构位于 细胞色素bc1或b6f复合体的一个亚单位的原子能级。 细胞色素f具有c-型的三个前所未有的结构特征。 (A)一个主要的β-链基序，(B)两个可区分的细胞色素 结构域，以及(C)作为轴的Tyr-1的N-末端的α-氨基 第六个亚铁血红素配体：后一个结果提供了关于 Cyt f跨膜移位中的一系列事件， 即加工必须先于完成亚铁血红素的协调和最终的 集合。(Ii)纯化的b6f络合物的结构特征为 和功能性二聚体。(Iii)b6f复合体的螺旋间力为 被发现是相对薄弱的。(Iv)β-亚基的定位 研究发现，血红素交联的细胞色素b-559与细胞色素b-559平行 α与(A)它是杂二聚体的预测一致， (B)膜蛋白的顺式正定向规则；及。(C) α和β的偶极势能的计算贡献 螺旋上升到其非常正的中点电位。 建议：(I)使用细胞色素f结构和现有的交叉连接 以链接信息为基础的“智能”站点导向 突变以(A)确定对接地点(S)的位置 以及(B)制造一组单一组氨酸表面突变体 在用Ru(Bpy)2加合物修饰后，将用来测量 与之相关的重组能量、伽马和最佳途径 蛋白质内的电子转移。据推测，这一转移将 有一个异常小的伽马，因为转移到塑料蓝蛋白 受体是等电势的。(C)组装Cyt f和Cat的后果 将对复合体的亚单位进行加工抑制试验 和释放Tyr-1氨基。(Ii)a)均分散MR 将使用230,000个b6f二聚体来结晶该整体膜。 蛋白质复合体。(B)将测试二聚体b6f络合物的功能 在涉及N侧激酶的跨膜信号中，(C) Cyt b6高度保守的N侧外在环在细胞周期调控中的作用 外周蛋白质的对接。(Iii)地形的影响 细胞色素b-559血红素的反转将通过应用顺式- 正性规则及其跨膜分布的反转 带电的氨基酸。

The highly conserved cytochrome b6f complex and the reaction center cytochrome b-559 of oxygenic photosynthesis will be used to study basic aspects of the structure and function of integral membrane cytochromes. Thr proposed studies are based on the following results from previous grant support: (i) the solution of the crystal structure at 2.3A resolution of the active major extrinsic domain of cytochrome f, the first structure at the atomic level of a subunit of the cytochrome bc1 or b6f complexes. Cytochrome f has three unprecedented structural features for a c-type cytochrome of (a) a predominant beta-strand motif, (b) two distinguishable domains, and (c) the N-terminal alpha-amino group of Tyr-1 as the axial sixth heme ligand: the latter result provided specific information about the sequence of events in the translocation of cyt f across the membrane, i.e. that processing must precede completion of heme coordination and final assembly. (ii) The purified b6f complex was characterized as a structural and functional dimer. (iii) The interhelix forces of the b6f complex were found to be relatively weak. (iv) The orientation of the beta-subunit of that heme cross-linked cytochrome b-559 was found to be parallel to that of the alpha, in agreement with (a) the prediction that it is a heterodimer, (b) the cis-positive rule for orientation of membrane proteins, and (c) the calculated contribution of the dipole potential of the alpha and beta helices to its very positive midpoint potential. It is proposed; (I) to use the cytochrome f structure and existing cross- linking information as the basis for 'intelligent' site-directed mutagenesis to (a) determine the position of the docking site(s) for plastocyanin, and (b) to make a set of single histidine surface mutants that will be utilized, after modification wit Ru (bpy)2 adducts, to measure the reorganization energy, gamma, and optimum pathway associated with the intraprotein electron transfer. It is hypothesized that this transfer will have an unusually small gamma because the transfer to the plastocyanin acceptor is isopotential. (c) The consequences for assembly of cyt f and the subunits of the complex will be tested of inhibition of the processing and liberation of the Tyr-1 amino group. (II,a) The homodisperse Mr 230,000 b6f dimer will be used to crystallize this integral membrane protein complex. (b) The function of dimeric b6f complex will be tested in trans-membrane signaling involving the n-side kinase, as will (c) the role of the very highly conserved n-side extrinsic loops of cyt b6 in the docking of peripheral proteins. (iii) The effect of topographical inversion of the cyt b-559 heme will be examined by applying the cis- positive rule and reversing its trans-membrane distribution of positively charged amino acids.