Problems in Membrane Protein Crystallography: Hetero-Oligomeric Cytochrome b6f

描述（由申请人提供）：异源寡聚细胞色素b6 f和bc 1复合物位于光合和呼吸能量转换膜中电子转移链的中心。这种膜含有相对较少的异源寡聚整合膜蛋白中的大多数，这些蛋白已被X射线晶体学解析到分辨率d 3.0 E。对二聚体220 kDa八亚基整合b6 f复合物的结晶的研究将分析与整合膜蛋白的结晶一般相关的蛋白水解和脂质-蛋白质相互作用的问题。结构-功能分析将集中在一个独特的氧化还原基团，血红素cn的性质，并在醌（ol）跨膜转移的机制，携带电子和质子，并被血红素cn还原。拟开展的研究：（1）晶体制备;蛋白水解。B6 F复合物不能从可转化的单细胞蓝细菌中分离，因为B6 F二聚体是单体化的，并且在从膜中提取时变得无活性和不可结晶。蓝藻b6 f的成功结晶利用了丝状M. laminosus，其中蛋白水解的程度较小。然而，M.板层肌不能变形。因此，b6 f复合物的蓝藻来源将改变为丝状念珠藻（鱼腥藻）sp. PCC 7120，从其获得活性和可结晶的复合物。由于这种蛋白质水解问题经常阻碍膜蛋白结晶的努力，单细胞蓝藻中的关键蛋白酶将通过质谱和蛋白质组学分析来鉴定。(2)磷脂的功能。蛋白质内脂质的功能仅在少数多亚基膜蛋白中进行了分析。我们的新的脂质增加程序导致结晶速率的大幅增加和晶体质量的改善。植物类囊体膜b6 f复合物，其中含有第九（FNR）亚基，其结晶取决于独特的不同（阴离子DOPG）脂质的晶体的性质，正在研究中，是依赖于电子转移活性和结晶速率的性质添加脂质。(3)血红素cn的功能; b6 f复合体的进化。独特的血红素CN，没有发现在泛醌含细胞色素Bc 1复合物的功能，将研究在念珠藻定点诱变，并通过结构-功能分析，在厚壁菌，如枯草芽孢杆菌，在遗传学上接近蓝藻。组氨酸标记的，启动子增强的厚壁菌“qcr”复合物将被纯化和筛选，用于与甲基萘醌的结晶和电子转移反应。(4)醌通过狭窄的p侧入口转移。电子和质子通过亲脂性ubi-和plastoquinone（PQ）携带穿过bc 1和b6 f复合物中的单体间醌交换腔。PQ/PQH 2发现，进入和退出一个狭窄的11 × 12 E p-侧门户的机制将通过门户残基的诱变和门户力场的计算分析进行研究。公共卫生相关性：这些研究的一些生物医学相关方面是，它们旨在了解介导所有跨生物膜运输（包括营养素和药物）的蛋白质的详细内部结构。通过细胞膜，这组能量转换蛋白决定了人类细胞中的能量水平及其调节。

DESCRIPTION (provided by applicant): The hetero-oligomeric cytochrome b6f and bc1 complexes are in the center of the electron transfer chains in photosynthetic and respiratory energy transducing membranes. Such membranes contain the majority of the relatively few hetero-oligomeric integral membrane proteins that have been solved by X-ray crystallography to a resolution d 3.0 E. Studies on crystallization of the dimeric 220 kDa eight subunit integral b6f complex would analyze problems of proteolysis and lipid-protein interactions that are of general relevance to the crystallization of integral membrane proteins. Structure-function analysis would focus on the properties of a unique redox group, heme cn, and on the mechanism of transfer across the membrane of quinone (ol) that carries the electrons and protons and is reduced by heme cn. Proposed studies: (1) Crystal preparation; proteolysis. b6f complex cannot be isolated from transformable unicellular cyanobacteria because the b6f dimer is monomerized and rendered inactive and non-crystallizable upon extraction from the membrane. Successful crystallization of cyanobacterial b6f has utilized the filamentous M. laminosus, in which the extent of proteolysis is smaller. However, M. laminosus is not transformable. Therefore, the cyanobacterial source of b6f complex will be changed to the filamentous Nostoc (Anabaena) sp. PCC 7120, from which active and crystallizable complex has been obtained. Because this kind of proteolysis problem frequently hinders efforts to crystallize membrane proteins, the critical protease(s) in the unicellular cyanobacteria would be identified by mass spectroscopic and proteomic analysis. (2) Function of phospholipids. The function of intra-protein lipids has been analyzed in only a few multi-subunit membrane proteins. Our novel lipid augmentation procedure resulted in a major increase in the rate of crystallization and improvement in crystal quality. The properties of crystals of the plant thylakoid membrane b6f complex, which contains a ninth (FNR) subunit and whose crystallization depends uniquely on a different (anionic DOPG) lipid, is under study, as is the dependence of electron transfer activity and rate of crystallization on the nature of added lipids. (3) Functions of heme cn; evolution of b6f complex. The function of the unique heme cn, not found in ubiquinone-containing cyt bc1 complexes, will be studied by site-directed mutagenesis in Nostoc and, through structure-function analysis, in firmicutes such as Bacillus subtilis that are phylogenetically close to cyanobacteria. His-tagged, promoter-augmented firmicute "qcr" complex will be purified and screened for crystallization and electron transfer reactions with menaquinone. (4) Quinone transfer though the narrow p-side portal. Electrons and protons are carried across an inter-monomer quinone exchange cavity in bc1 and b6f complexes by lipophilic ubi- and plastoquinone (PQ). The mechanisms by which PQ/PQH2 finds, enters, and exits a narrow 11 x 12 E p-side portal will be studied through mutagenesis of portal residues and computational analysis of the portal force field. PUBLIC HEALTH RELEVANCE: Some of the biomedically relevant aspects of these studies are that they are directed toward an understanding of the detailed internal structure of the proteins that mediate all traffic, including nutrients and drugs, across biological membranes. Via the membrane, the set of energy-transducing proteins determines the level of energy and its regulation in the human cell.