Binding Protein Medicated Membrane Transport Systems

结合蛋白介导的核糖膜转运系统在大肠杆菌中的结构和功能将通过两种主要方法进行探讨。首先，将继续对核糖结合蛋白进行定点诱变研究，以确定核糖结合蛋白上与趋化性（trg）和细胞质膜上的核糖转运受体相互作用的表面。我们最近对这种类型的研究发现，当一个Ile残基变成Arg时，在结合蛋白的“铰链”附近有一个位点消除了趋化性。计划进一步探测该位点及其附近的残基，以及结合蛋白裂口附近似乎影响转运的位点。其次，核糖膜转运复合物组分的过表达、分离和表征将继续进行。该复合体的三个蛋白中有两个已被纯化，其中一个已被证明与一个atp结合蛋白家族同源，包括真核多重耐药蛋白和新发现的囊性纤维化相关蛋白。将对纯化的转运蛋白进行物理和生化研究，并继续尝试过表达第三种转运蛋白。一旦这三种转运蛋白都产生，将在体外进行转运蛋白的重组。一个重组的系统，以及与转运蛋白相互作用的结合蛋白表面相关的信息，将允许未来对转运机制的生化研究。一个相关的目标是过度表达rbs操纵子的抑制因子。一种被证明既与抑制蛋白家族同源又与核糖结合蛋白所属的结合蛋白家族同源的分子这导致了一个抑制机制的模型，一旦获得大量的纯抑制物，该模型将在生物化学和结构上进行测试。

The structure and function of the components of the binding protein mediated membrane transport system for ribose in E. coli will be probed by two main approaches. First, site-directed mutagenesis studies on the ribose binding protein will be continued in order to define the surfaces on the ribose binding protein which interact with the chemotaxis (trg) and the ribose transport receptors on the cytoplasmic membrane. Our recant studies of this type located one site near the "hinge" of the binding protein which eliminated chemotaxis when an Ile residue was changed to Arg. Further probing of this site and residues near it are planned as well as sites near the cleft of the binding protein which appear to affect transport. Second, overexpression, isolation, and characterization of the components of the membrane transport complex for ribose will be continued. Two of the three proteins in the complex have been purified, one of which has been shown to be homologous to a family of ATP-binding proteins including the eukaryotic multiple drug resistance proteins and the newly identified cystic fibrosis-related protein. Physical and biochemical studies will be performed on the purified transport protein, and attempts to overexpress the third will be continued. Once all three are produced, reconstitution of the transporter in vitro will be pursued. A reconstituted system, along with the information related to the surface of the binding protein which interacts with the transported, will allow future biochemical studies of the mechanism of transport, A related objective is to overexpress the repressor for the rbs operon, a molecule which was shown to be homologous both to a family of repressors and also to the family of binding proteins to which ribose binding protein belongs. This led to a model for the repressor mechanism which will be testable biochemically and structurally once significant amounts of the pure repressor are obtained.