MEMBRANE DOMAINS OF A NOVEL MG++ ATPASE--GENETIC APPROACHES TO P-CLASS ATPASES

新型 MG ATP 酶的膜域——P 类 ATP 酶的遗传方法

• 批准号: 6272553
• 负责人: MICHAEL E MAGUIRE
• 金额: $ 17.87万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6272553
• 关键词: Salmonella typhimurium; active sites; adenosinetriphosphatase; calcium transporting ATPase; conformation; enzyme mechanism; gene deletion mutation; genetic promoter element; ion transport; isozymes; magnesium; membrane structure; protein structure function; site directed mutagenesis; synthetic enzyme

MgtB is a P-type ATPase of Salmonella typhimurium that mediates the influx of Mg2+ against its electrochemical gradient, has minimal homology to known prokaryotic P-type ATPases and is most homologous to mammalian Ca2+-ATPases thus making it an excellent model of sarcoplasmic reticular Ca2+-ATPases. Because the molecular genetic techniques available in prokaryotes are easier, faster and in many cases more advanced than in eukaryotic systems, important experimental approaches are available for study of the structure of membrane proteins that are not feasible in eukaryotes. We propose to examine the membrane domain of MgtB using an iterative process combining cysteine scanning mutagenesis and crosslinking with molecular modeling to provide an enhanced structure of the membrane domain. The end result with be a molecularly detailed picture of the spatial orientation of each transmembrane segment to every other segment. Succinctly, we will determine for each transmembrane segment "which face faces which face." The following specific questions will be asked: 1) What is the structure of the membrane domain of a P-type ATPase? For a given transmembrane segment of MgtB, we will determine which other transmembrane segments are immediately adjacent to allow construction of a medium resolution model of the membrane domain of a P-type ATPase. A "library" of mutant MgtB isoforms will be created by mutagenizing selected residues within membrane domains to cysteine; double mutants will be then constructed with cysteine substitutions in two different membrane domains. Subsequent oxidation will form disulfide bonds and crosslink only those cysteines with appropriately close spatial orientations within the membrane. This approach is only feasible in a prokaryotic cell. 2) Which membrane domains shift position during the reaction cycle of a P-type ATPase? The evolving membrane domain model will allow prediction of specific amino acid residues within neighboring helices whose relative positions may shift, via rotation and/or translation, during the phosphorylation-dephosphorylation cycle of a P-type ATPase. The predictions will be tested by determining the efficiency of cysteine crosslinking between selected pairs of residues as a function of the enzyme's phosphorylation state. 3) What changes occur in metal binding sites during the reaction cycle of a P-type ATPase? MgtB will be purified using a 6xHis tag. In collaboration with Dr. J.K. Blasie, purified, detergent-solubilized enzyme will be covalently tethered via an extracytoplasmic cysteine to the sulfhydryl endgroup surface of an organic self-assembled monolayer chemisorbed onto the surface of a Ge/Si multilayer substrate. This will provide vectorially oriented enzyme to determine the profile structure of MgtB using nonresonance x-ray diffraction, thereby more firmly establishing its membrane domain structure. Within this profile structure, using resonance x-ray diffraction, the distribution of bound Ni2+, as surrogate for Mg2+ on the enzyme's high-affinity sites will then be determined.

MGTB是鼠伤寒沙门氏菌的P型ATPase，可介导涌入 与其电化学梯度相对的MG2+，与已知的同源性最小 原核P型ATPases，最与哺乳动物Ca2+-ATPase同源 从而使其成为核质网Ca2+-ATPase的出色模型。 因为原核生物中可用的分子遗传技术是 比真核系统更容易，更快，在许多情况下， 重要的实验方法可用于研究结构 在真核生物中不可行的膜蛋白。 我们建议 使用迭代过程组合检查MGTB的膜结构域 半胱氨酸扫描诱变和与分子建模的交联 提供膜结构域的增强结构。 最终结果与 成为每个分子的详细图片 将跨膜段转换为其他每个细分市场。 简洁地，我们会的 确定每个跨膜段“面部面部的面部”。 将问以下特定问题：1）结构是什么 P型ATPase的膜结构域？ 对于给定的跨膜 MGTB的段，我们将确定哪些其他跨膜段是 立即邻近以构建中等分辨率的模型 P型ATPase的膜结构域。 突变MGTB的“库” 同工型将通过诱变膜内的残基而产生 半胱氨酸的域；然后将用半胱氨酸构建双突变体 在两个不同的膜结构域中取代。 随后的氧化会 形成二硫键，并仅与适当的半胱氨酸交联 关闭膜内的空间取向。 这种方法只是 在核细胞中可行。 2）哪些膜域移动位置 在P型ATPase的反应周期中？ 不断发展的膜域 模型将允许预测特定的氨基酸残基 相对螺旋的相对位置可能通过旋转和/或 翻译，在P型的磷酸化 - 二磷酸化周期 ATPase。 这些预测将通过确定的效率来测试 选定的残基对之间的半胱氨酸交联作为 酶的磷酸化状态。 3）金属中发生了什么变化 在P型ATPase的反应周期中结合位点？ MGTB会 使用6xHis标签纯化。 与J.K.博士合作布拉西， 纯化的洗涤剂 - 溶解酶将通过一个共价束缚 胞质半胱氨酸到有机的硫二酰端基表面 自组装的单层化学吸附在GE/SI多层的表面上 基材。 这将提供以矢量为导向的酶来确定 使用非共振X射线衍射的MGTB的配置文件结构，从而 更牢固地建立其膜域结构。 在此 曲线结构，使用共振X射线衍射，分布 绑定的ni2+，作为MG2+的替代酶的高亲和力地点 然后确定。