BINDING PROTEINS FOR ACTIVE TRANSPORT & CHEMOTAXIS

对周质结合作用的分子理解 细菌中活跃的运输和趋化作用中的蛋白质需要 这一蛋白质家族的详细图片。我们的长期目标是 确定几种结合的结构和功能 作为运输和运输的初始受体的蛋白质 趋化性是两个重要的生物学过程。因此，一个x- 七种不同结合蛋白-AT的射线结构分析 每个主要群体中至少有两人具有特定的 碳水化合物、无机阴离子和氨基酸-一直是 已启动。这一目标是可以实现的，因为在 解算六种结合蛋白的三维结构 L专用-阿拉伯糖、D-半乳糖、D-麦芽糖、硫酸盐、 亮氨酸/异亮氨酸/缬氨酸和亮氨酸是最近实现的。 磷酸盐结合蛋白最近已经结晶。 这些结构分析还揭示了 糖、硫酸盐阴离子和氨基酸的结合方式 Zwitterion。拟议研究的主要目标是将 四种糖类结合蛋白的结构分析 一种解决磷酸盐结合结构的方法 蛋白。不同糖底物与糖的结合 衍生品将通过高分辨率结构进行详细研究 分析。其他相关研究也将展开。这个 每个糖羟基相互作用对整体的贡献 蛋白质-糖复合体的亲和力将用不同的方法进行评估 以脱氧/氟取代的单吡喃糖苷为探针。约束： 这些探针将通过结晶学、平衡学和 动力学结合和理论技术。有了这三个- 立体结构提供坚实的基础，独一无二 结合蛋白的结构和功能特征将是 通过定点突变进一步探索产生 在原代中具有特定改变的蛋白质 蛋白质的序列。产生的突变蛋白将是 分离并广泛表现出关于他们的 结合活性、物理和化学性质。的结构 突变株也将被确定为直接与 母体野生型结构。

A molecular understanding of the role of periplasmic binding proteins in active transport and chemotaxis in bacteria requires a detailed pictures of this family of proteins. Our long range goal is to determine the structure and function of several binding proteins which serve as initial receptors for transport and chemotaxis, two important biological process. Accordingly, an x- ray structural analysis of seven different binding proteins - at least two from each of the major groups with specificity for carbohydrates, inorganic anions, and amino acids - has been initiated. This goal is attainable because considerable success in solving the three-dimensional structures of six binding proteins specific for L-arabinose, D-galactose, D-maltose, sulfate, leucine/isoleucine/valine and leucine has recently been achieved. The phosphate-binding protein has recently been crystallized. These structural analyses have also revealed novel features of the mode of binding of sugars, sulfate anion and amino acid zwitterion. The major goal of the proposed research is extend the structural analysis of four binding proteins to better than 2 sugar A resolution and the solve the structure of the phosphate - binding protein. The binding of different sugar substrates and sugar derivatives will be examined in detail by high resolution structure analysis. Other correlative studies will also be undertaken. The contribution of each sugar hydroxyl interaction to the overall affinity of protein-sugar complexes will be assessed with various deoxy/fluoro substituted monopyranosides as probes. Binding of these probes will be assessed by crystallographic, equilibrium and kinetic binding, and theorteical techniques. With the three- dimensional structures providing a solid foundation, unique structural and functional features in the binding proteins will be further explored by site-directed mutagenesis to generate proteins with specific alterations at desired points in the primary sequence of the protein. Mutant proteins produced will be isolated and characterized extensively with regard to their binding activity, physical and chemical properties. Structures of mutants will also be determined for direct comparison with the parent wild-type structures.