Problems in Membrane Protein Crystallography: Hetero-Oligomeric Cytochrome b6f

描述(申请人提供)：异寡聚体细胞色素b6f和bc1复合体位于光合作用和呼吸能量转导膜电子传递链的中心。这种膜含有相对较少的异寡聚完整膜蛋白的大部分，这些蛋白已经通过X射线结晶学解析到d 3.0E。对二聚体220 kDa八亚单位完整b6f复合体的结晶研究将分析与完整膜蛋白结晶普遍相关的蛋白质分解和脂-蛋白相互作用问题。结构-功能分析将侧重于一个独特的氧化还原基团--血红素CN的性质，以及携带电子和质子并被血红素CN还原的苯醌(Ol)的跨膜转移机制。建议的研究：(1)晶体制备；蛋白质分解。B6f复合体不能从可转化的单细胞蓝藻中分离出来，因为b6f二聚体是单体，从膜上提取后变得不活跃和不结晶。蓝藻B6f的成功结晶利用了丝状的层状分枝杆菌，其中蛋白质的降解程度较小。然而，Laminosus是不能变形的。因此，b6f复合体的蓝藻来源将改为丝状发菜(Anabaena)sp.PCC7120，从中得到活性的、可结晶的络合物。由于这种蛋白分解问题经常阻碍膜蛋白结晶的努力，因此可以通过质谱学和蛋白质组学分析来鉴定单细胞蓝藻中的关键蛋白酶(S)。(2)磷脂的功能。仅在少数多亚单位膜蛋白中分析了蛋白内脂类的功能。我们的新的脂质增强过程导致了结晶速度的显著提高和晶体质量的改善。植物类囊体膜b6f复合体的晶体性质正在研究中，该复合体含有第九个(FNR)亚单位，其结晶唯一地依赖于不同的(阴离子DOPG)脂类，以及电子转移活性和结晶速度对添加脂类的性质的依赖。(3)血红素CN的功能；b6f复合体的进化。独特的血红素CN在含有泛醌的细胞色素Bc1复合体中没有发现，它的功能将通过发菜的定点突变来研究，并通过结构功能分析，在系统发育上与蓝藻接近的枯草芽孢杆菌等坚果中进行研究。His标记的、启动子增强的FirmicutQCR复合体将被提纯，并筛选结晶和与孟喹酮的电子转移反应。(4)Quinone通过狭窄的p侧门转移。在bc1和b6f络合物中，电子和质子通过亲脂性的ubi和plastoquone(PQ)穿过单体间的苯醌交换空腔。PQ/PQH2发现、进入和退出狭窄的11×12 Ep侧入口的机制将通过入口残基的诱变和入口力场的计算分析来研究。与公共健康相关：这些研究的一些生物医学相关方面是，它们针对的是对蛋白质的详细内部结构的理解，这些蛋白质调节所有跨越生物膜的运输，包括营养和药物。通过细胞膜，这组能量转导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.