Specificity/Regulation of Cyanobacterial ABC Transporters

描述（由申请人提供）：我们大气中温室气体的积累已经并将继续导致全球气温稳步上升。阻止温室气体，特别是二氧化碳在大气中进一步积累的一种方法是通过操纵水生蓝藻的天然碳螯合机制。限制这些生物体中碳螯合的一个重要因素是铁的低生物利用度。为了获得碳酸氢盐和铁，蓝藻采用高亲和力ATP结合盒（ABC）转运蛋白。这些ABC转运蛋白由一个周质溶质结合脂蛋白、一个跨细胞质膜通透酶和一个为溶质转运提供动力的ATP酶组成。X-射线晶体学，生物化学和分子生物学技术的组合将被用来检查在碳酸氢盐CmpABCD和集胞藻属PCC 6803的三价铁FutABC转运蛋白的底物特异性和运输调节的分子基础。将追求的CmpA，CmpC和CmpD蛋白的X射线晶体结构，以及独特的多结构域CmpC蛋白的生物化学表征，参与碳酸氢盐摄取的调节。热力学和酶的技术将被用来研究CmpC和CmpD的ATP酶结构域之间的相互作用。FutABC转运蛋白将被用来询问ABC转运蛋白的膜透性酶和ATP酶组分是否在溶质识别中起作用的问题。铁结合蛋白FutA 1的溶质结合特异性将被改变，以测试FutBC机器是否可以接受替代金属离子。这些实验将涉及集胞藻的遗传操作，目的是通过FutBC测试金属离子的获取。

DESCRIPTION (provided by applicant): The build-up of greenhouse gases in our atmosphere has, and will continue to, lead to a steady increase in the global temperature. A way to halt the further atmospheric accumulation of greenhouses gases, particularly carbon dioxide, is through manipulation of the natural carbon-sequestering mechanisms of aquatic cyanobacteria. One significant factor limiting carbon sequestration in these organisms is the low bioavailability of iron. For the acquisition of bicarbonate and iron, cyanobacteria employ high-affinity ATP-binding cassette (ABC) transporters. These ABC transporters are composed of a periplasmic solute-binding lipoprotein, a cytoplasmic-membrane spanning permease, and an ATPase that powers solute transport. A combination of x-ray crystallography, biochemical and molecular biology techniques will be used to examine both the molecular basis of substrate specificity and transport regulation in the bicarbonate CmpABCD, and the ferric iron FutABC transporters of Synechocystis sp PCC 6803. X-ray crystal structures of the CmpA, CmpC and CmpD proteins will be pursued, as well as a biochemical characterization of the unique multi-domain CmpC protein that is involved in the regulation of bicarbonate uptake. Both thermodynamic and enzymatic techniques will be employed to investigate the interactions between the ATPase domains of CmpC and CmpD. The FutABC transporter will be used to ask the question of whether the membrane permease and ATPase components of ABC transporters plays a role in solute recognition. The solute-binding specificity of the ferric-binding protein FutA1 will be altered in order to test whether the FutBC machinery can accept alternate metal ions. These experiments will involve genetic manipulation of Synechocystis for the purpose of testing metal ion acquisition through FutBC.