BINDING PROTEINS FOR ACTIVE TRANSPORT AND CHEMOTAXIS

主动运输和趋化性的结合蛋白

• 批准号: 3270454
• 负责人: FLORANTE A QUIOCHO
• 金额: $ 12.71万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-12-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270454
• 关键词: X ray crystallography; active transport; aminoacid transport; arabinose; bacteria; bacterial proteins; binding proteins; carbohydrate transport; chemotaxis; galactose; genetic manipulation; isoleucine; leucine; maltose; phosphates; point mutation; protein engineering; protein structure function; receptor; site directed mutagenesis; stop flow technique; sulfates; transport proteins; valine

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.

对周质结合作用的分子理解 蛋白质在细菌中的主动运输和趋化性需要 这个蛋白质家族的详细图片。 我们的长远目标是 确定几种结合的结构和功能， 作为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，x- 七种不同结合蛋白的X射线结构分析-- 每个主要群体中至少有两个具有特异性， 碳水化合物，无机阴离子和氨基酸-已经被 启动。 这一目标是可以实现的，因为在 解析六种结合蛋白的三维结构 对L-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。 磷酸盐结合蛋白最近已结晶。 这些结构分析也揭示了新的特点， 糖、硫酸根阴离子和氨基酸的结合模式 [咒语] 本研究的主要目标是将 四种优于2糖结合蛋白结构分析 一种解决和解决磷酸结合结构的方法 蛋白 不同糖底物与糖的结合 衍生物将通过高分辨率结构详细检查 分析. 还将开展其他相关研究。 的 每种糖羟基相互作用对总体 蛋白质-糖复合物的亲和性将用各种方法来评估。 脱氧/氟取代的单吡喃糖苷作为探针。 结合 这些探针将通过晶体学、平衡和 动力学结合和理论技术。 有了这三个- 三维结构提供了坚实的基础，独特的 结合蛋白的结构和功能特征将被 通过定点诱变进一步探索以产生 在初级细胞中的期望点具有特定改变的蛋白质 蛋白质的序列。 产生的突变蛋白质将 孤立和广泛的特点，关于他们的 结合活性、物理和化学性质。 结构 突变体也将被确定用于与 亲本野生型结构。