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

MgtB是鼠伤寒沙门氏菌的一种P型ATPase，介导内流 相对于它的电化学梯度，与已知的同源性最小 原核的P型ATPase，与哺乳动物的Ca2+-ATPase同源性最高 从而使其成为肌浆网钙-三磷酸腺苷酶的极佳模型。 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，而且在许多情况下更先进， 重要的实验方法可用于结构的研究。 在真核生物中不可行的膜蛋白。我们建议 用迭代结合的方法研究镁铁蛋白的膜结构域 半胱氨酸扫描诱变和分子模拟交联技术研究 提供膜结构域的增强结构。最终结果是 是每个星体空间方位的分子细节图 跨膜节段到每隔一个节段。简而言之，我们将 确定每个跨膜段的“哪一面朝哪一面”。 将提出以下具体问题：1)结构是什么 P型ATPase的膜结构域？对于给定的跨膜 我们将确定哪些其他跨膜片段是 紧邻，允许建造中分辨率模型 P型ATPase的膜结构域。突变的mgtB的“文库” 异构体将通过诱变膜内选定的残基来产生 结构域到半胱氨酸；然后用半胱氨酸构建双突变体 两个不同膜结构域中的取代。随后的氧化将 形成二硫键，并只将半胱氨酸与适当的 膜内紧密的空间取向。此方法仅用于 在原核细胞中是可行的。2)哪些膜结构域位移 在P型ATPase的反应周期中？进化中的膜结构域 模型将允许预测特定的氨基酸残基 相对位置可通过旋转和/或移动的相邻螺旋 翻译，在P型蛋白的磷酸化-去磷酸化循环中 ATPase。这些预测将通过确定 所选残基对之间的半胱氨酸交联 酶的磷酸化状态。3)金属发生了哪些变化 P型ATPase反应周期中的结合部位？MGTB将 使用6xHis标签进行纯化。与J.K.布莱西博士合作， 纯化的、洗涤剂可溶解的酶将通过一种 胞外半胱氨酸到有机化合物的巯基端基表面 自组装单分子膜在Ge/Si多层膜表面的化学吸附 底物。这将提供面向载体的酶来确定 用非共振X射线衍射法研究了MgTB的轮廓结构，从而 更牢固地建立其膜结构域结构。在这个范围内 剖面结构，用共振x射线衍射法测定了其分布 结合Ni2+，作为酶的高亲和力位点上的镁离子的替代品，将 那就下定决心吧。

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.