Structural Studies of Metabolic Membrane Proteins

描述(申请人提供)：我们已经获得了参与细菌甘油代谢途径的两种膜蛋白的衍射晶，甘油促进剂和甘油-3-磷酸脱氢酶。这是确定晶体结构的重要一步，这些结构将产生有价值的新见解，将蛋白质折叠与功能和蛋白质-蛋白质相互作用以及催化、调节和跨膜吸收溶质分子的机制联系起来。细菌增殖的潜在有效机制是调节氧化和碳水化合物代谢的手段。这项结构研究集中在阐明关键的细菌膜蛋白的结构-功能关系，这些蛋白介导了基本的氧化和碳水化合物代谢。我们从事这些代谢蛋白的结构研究已经有几年了，现在正处于将我们的所有结果整合到这些致病细菌的代谢的全面和连贯的图景中的关键时刻。我们已经确定了氧化和甘油代谢途径的另外三个成员的结构，包括NADH过氧化物酶、NADH氧化酶和甘油激酶。这项提案解决了结构与职能和监管相关的关键问题。甘氨酸-3-磷酸脱氢酶具有特别重要的医学意义，因为它是为形成生物膜的多糖的生物合成提供磷酸三糖中间体的关键角色，并保护细菌免受脱水和抗生素治疗。这种膜-蛋白质结构研究有可能产生高通量结构基因组学无法提供的新的和新的结果。通过从革兰氏阳性肺炎链球菌获得甘油促进剂的衍射晶体(2.4A)，我们已经克服了膜蛋白结构研究中一些最困难和最限速的障碍。革兰氏阳性肺炎链球菌是一种参与甘油摄取的膜蛋白，很可能通过与甘油激酶的蛋白质-蛋白质相互作用来调节，我们最近确定了甘油激酶的结构。这种细菌与革兰氏阴性大肠埃希菌之间存在细微但显著的差异。我们得到了来自铜绿假单胞菌的甘油-3-磷酸脱氢酶(3.3A)的衍射晶体，并有希望获得更好的衍射晶体。我们的具体目标是获得这两种膜蛋白的原子分辨结构，这两种结构都可能成为抗生素设计和治疗的新靶点。

DESCRIPTION (provided by applicant): We have obtained diffracting crystals of two membrane proteins involved in the glycerol metabolic pathway in bacteria, glycerol facilitator and glycerol-3-phosphate dehydrogenase. This is a significant step towards crystal structure determination and these structures will yield valuable new insight, linking protein folds to function and protein-protein interactions as well as mechanisms of catalysis, regulation, and transmembrane uptake of solute molecules. Underlying effective mechanisms of bacterial proliferation are means of mediating oxidative and carbohydrate metabolism. This structural study focuses on elucidating structure-function relationships of key bacterial membrane proteins that mediate fundamental oxidative and carbohydrate metabolism. We have been engaged in structural studies of these metabolic proteins for several years and are at pivotal point in integrating all of our results into a comprehensive and coherent picture of metabolism in these pathogenic bacteria. We have determined the structures of three other members of the oxidative and glycerol metabolism pathways, including NADH peroxidase, NADH oxidase, and glycerol kinase. This proposal addresses key questions correlating structure to function and regulation. Gly-3-phosphate dehydrogenase is of particular medical importance as it is a key player in providing triose phosphate intermediates for biosynthesis of polysaccharides that form biofilm and protects the bacterium from dehydration as well as antibiotic therapy. This membrane-protein structural study has the potential to yield new and novel results, which are not provided by high-throughput structural genomics. We have overcome some of the most difficult and rate-limiting hurdles in membrane protein structural studies by obtained diffracting crystals of the glycerol facilitator (2.4 A) from Gram-positive Streptococcus pneumonia, a membrane protein involved in glycerol uptake and likely to be regulated via protein-protein interactions with glycerol kinase, whose structure we've recently determined. Subtle but significant differences exist between this and the Gram-negative E. coli facilitator. We have diffracting crystals of the glycerol-3-phosphate dehydrogenase (3.3 A) from Pseudomonas aeruginosa and have promising indications for better diffracting crystals. Our specific aims are to obtain atomic resolution structures of both membrane proteins, both of which are likely to be novel targets for antibiotic design and therapy.