ERYTHROCYTE MEMBRANE CYTOSKELETON ASSOCIATIONS

描述：（改编自研究者摘要）在本提案中， 首席研究员打算做详细的分子生物学和 人红细胞锚蛋白和人红细胞的生化研究 内收蛋白 红细胞锚蛋白的研究将包括结构分析 89 kDa结构域及其组成的33个残基重复序列。 定义 89 kDa结构域的区域将在细菌中表达， 天然二级结构所需的重复次数，通过 将测定圆二色性。 89 k Da结构域的晶体将 制备用于通过X射线衍射分析结构。 的结合 红细胞带3的33个残基重复区域内的位点， 域将被映射，并且重复和独特重复的最小数目 所需的约束力确定。 微管蛋白和Na/K位点的定位 将进行89 k Da结构域内的ATP酶结合。 结构域 其它具有同源33个残基重复的蛋白质（DrosophilaNotch和C. elegans lin 12基因产物）将被表达，并且它们将被用于 鉴定微管蛋白和其它蛋白可能共同结合特异性 具有周期性结构的大分子（核酸、磷脂）。 将绘制红细胞和脑锚蛋白的血影蛋白结合位点 和能够表达血影蛋白结合的最小区域 活动确定。 这些锚蛋白中提供 对红细胞血影蛋白的特异性，以及对脑血影蛋白的脑锚蛋白的特异性， 将被确定。 锚蛋白缺失区域可能是一个新的假说， 蛋白质2.2是mRNA选择性剪接的产物，其功能是 将探索假底物和阻断剂的潜在结合位点。 肾微粒体中的蛋白质已被证明可以识别 将鉴定蛋白2.2而不是未剪接的锚蛋白。 第二主 该研究的组成部分涉及人红细胞内收蛋白。 完成 人β亚基和aplha基因cDNA的克隆和序列分析 红细胞内收蛋白。 两种亚基都将表达， 重组以形成异二聚体和可能的同二聚体。 活性中心 与钙调蛋白、肌动蛋白、血影蛋白/肌动蛋白的相互作用， 异二聚体和异二聚体之间的缔合以形成四聚体， 高级低聚物将使用作图技术测定。 位点 还将测定蛋白激酶A和C的磷酸化。

DESCRIPTION: (Adapted from investigator's abstract) In this proposal, the principal investigator intends to do detailed molecular biology and biochemical studies of human erythrocyte ankyrin and human erythrocyte adducin. Studies on erythrocyte ankyrin will include structural analysis of the 89 kDa domain and its constituient 33-residue repeats. Defined regions of the 89 k Da domain will be expressed in bacteria and the minimal number of repeats required for native secondary structure as monitored by circular dichroism will be determined. Crystals of the 89 k Da domain will be prepared for analysis of structure by X-ray diffraction. The binding sites for erythrocyte band 3 within the 33 residue repeat region of this domain will be mapped and the minimal number of repeats and unique repeats required for binding determined. Mapping for sites of tubulin and Na/K ATPase binding within the 89 k Da domain will be performed. Domains of other proteins with homologous 33 residue repeats (Drosophila Notch and C. elegans lin12 gene products) will be expressed and they will be used to identify possible common binding specificity for tubulin and other macromolecules with periodic structures (nucleic acids, phospholipids). The spectrin binding sites of red cell and brain ankyrins will be mapped and the minimum regions competent for expressing spectrin-binding activities determined. The regions within these ankyrins that provide specificity for red cell spectrin, and brain ankyrin for brain spectrin, will be determined. The hypothesis that the region of ankyrin deleted in protein 2.2, a product of alternatively spliced mRNA, functions as a pseudosubstrate and blocks potential binding sites will be explored. Proteins in kidney microsomes that have been demonstrated to recognize protein 2.2 and not unspliced ankyrin will be identified. The second major component of the study relates to human erythrocyte adducin. Complete cloning and sequencing of cDNA encoding aplha and beta subunits of human erythrocyte adducin are proposed. Boths subunits will be expressed and recombined to form heterodimers and possibly homodimers. Active sites for interaction with calmodulin, actin, spectrin/actin, subunit interactions to heterodimers and associations between heterodimers to form tetramers and higher oligomers will be determined using mapping techniques. Sites of phosphorylation by protein kinases A and C will also be determined.