Problems in Membrane Protein Crystallography: Hetero-Oligomeric Cytochrome b6f

描述（由申请人提供）：异低聚细胞色素b6f和bc1复合物位于光合和呼吸能量转导膜的电子传递链的中心。这些膜包含了大多数相对较少的异寡聚整体膜蛋白，这些蛋白质已经通过x射线晶体学解决了分辨率为3.0 e的问题。对二聚体220 kDa 8亚基整体b6f复合物的结晶研究将分析蛋白质水解和脂质-蛋白质相互作用问题，这些问题与整体膜蛋白的结晶一般相关。结构-功能分析将侧重于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还原的醌（醇）跨膜转移的机制。拟进行的研究：(1)晶体制备；蛋白质水解。B6f复合物不能从可转化的单细胞蓝藻中分离出来，因为B6f二聚体在从膜中提取时是单体化的，并且变得无活性和不可结晶。蓝藻b6f的成功结晶利用了丝状M. laminosus，其中蛋白质水解程度较小。然而，M. laminosus是不可转化的。因此，b6f络合物的蓝藻来源将改变为丝状Nostoc (Anabaena) sp. PCC 7120，从中获得了活性和可结晶的络合物。由于这种蛋白质水解问题经常阻碍膜蛋白的结晶，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确定。(2)磷脂的作用。蛋白内脂的功能仅在少数多亚基膜蛋白中得到了分析。我们新颖的脂质增强程序导致结晶率和晶体质量的改善的主要增加。植物类囊体膜b6f复合物的晶体性质正在研究中，该复合物含有第九亚基（FNR），其结晶依赖于不同的（阴离子DOPG）脂质，以及电子转移活性和结晶速率对添加脂质的依赖。(3)血红素cn的功能；b6f复合物的演化。独特的血红素cn的功能，在含泛素的cyt bc1复合体中没有发现，将通过Nostoc的位点定向诱变研究，并通过结构-功能分析，在系统发育上接近蓝藻的厚壁菌门，如枯草芽孢杆菌中研究。his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结晶和与甲基萘醌的电子转移反应。(4)醌通过狭窄的p侧门静脉转移。在bc1和b6f配合物中，电子和质子通过亲脂性双醌和质体醌（PQ）进行单体间醌交换腔。PQ/PQH2发现、进入和退出狭窄的11 x 12 E - p侧传送门的机制将通过传送门残留物的诱变和传送门力场的计算分析来研究。公共卫生相关性：这些研究的一些生物医学相关方面是，它们旨在了解介导包括营养物质和药物在内的所有生物膜运输的蛋白质的详细内部结构。通过细胞膜，一组能量转导蛋白决定了人体细胞的能量水平及其调控。

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.