STRUCTURE/FUNCTION OF MEMBRANE BOUND CYTOCHROMES

高度保守的细胞色素b6 f复合物和反应中心 利用细胞色素B-559进行基础研究 的结构和功能方面的完整膜细胞色素。 拟议的研究是基于以下结果从以前的赠款 支持：（一）在2.3A分辨率下的晶体结构的解决方案， 细胞色素f的活性主要外在结构域， 细胞色素bc 1或b6 f复合物的亚基的原子水平。 细胞色素f有三个前所未有的结构特征， （a）主要β链基序，（B）两个可区分的 结构域，和（c）Tyr-1的N-末端α-氨基作为轴向结构域， 第六个血红素配体：后一个结果提供了关于 细胞色素F跨膜易位的事件顺序， 即，处理必须先于血红素配位的完成， 组装件. (ii)纯化的b6 f复合物被表征为结构稳定的。 和功能性二聚体。 (iii)b6 f复合物的螺旋间力为 被认为是相对较弱的。 (iv)β-亚基的取向 血红素交联的细胞色素B-559被发现与 α，与（a）预测它是异二聚体一致， (b)膜蛋白取向的顺式正规则，以及（c） 计算的α和β的偶极势的贡献 螺旋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- 链接信息作为“智能”网站导向的基础 诱变，以（a）确定对接位点的位置 质体蓝素，和（B）制备一组单组氨酸表面突变体 在用Ru（bpy）2加合物修饰后，将其用于测量 重组能量，γ，和最佳途径与 蛋白质内电子转移 据推测，这一转移将 有一个异常小的γ，因为转移到质体蓝素， 受体是等电位的。 (c)细胞色素f和 将测试复合物的亚基对加工的抑制 和Tyr-1氨基的释放。 (II，a）均分散Mr 230，000 B6 F二聚体将用于使该整体膜结晶 蛋白质复合物 (b)将测试二聚体b6 f复合物的功能。 在涉及n侧激酶的跨膜信号传导中，如（c） 细胞色素B6的非常高度保守的N-侧外部环在细胞色素B6的表达中的作用 外周蛋白的对接。 (iii)地形的影响 将通过应用顺式- 正性规则及其反转正性跨膜分布 带电氨基酸

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.