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

MgtB是鼠伤寒沙门氏菌的P型ATP酶，介导内流 的Mg 2+对它的电化学梯度，具有最小的同源性，以已知的 与哺乳动物Ca ~（2+）-ATP酶同源性最高 因此使其成为肌浆网Ca 2 +-ATP酶的优良模型。 因为原核生物中可用的分子遗传技术 更容易，更快，在许多情况下比真核系统更先进， 为研究这种结构提供了重要的实验手段 在真核生物中是不可行的。 我们建议 检查MgtB的膜域，使用迭代过程结合 半胱氨酸扫描诱变和交联与分子模拟， 提供膜域的增强结构。 最终结果是， 是每个分子空间取向的分子细节图， 跨膜片段与每隔一个片段。 简而言之，我们将 确定每个跨膜片段的“哪个面面对哪个面”。" 将提出以下具体问题：1）结构是什么 膜结构域的一部分 根据给定跨膜 MgtB片段，我们将确定哪些其他跨膜片段是 直接相邻，以允许构建 P型ATP酶的膜结构域。 突变MgtB的“文库” 将通过诱变膜内的选定残基产生同种型 结构域的半胱氨酸;双突变体将构建半胱氨酸 在两个不同的膜结构域中的取代。 随后的氧化将 形成二硫键并仅使那些半胱氨酸与适当的 在膜内紧密的空间取向。 这种方法只是 在原核细胞中是可行的 2)哪些膜结构域转移位置 在一个P型ATP酶的反应周期？ 进化中的膜结构域 模型将允许预测特定的氨基酸残基内 其相对位置可以通过旋转和/或 翻译，在磷酸化-去磷酸化周期的P-型 ATP酶 这些预测将通过确定 选择的残基对之间的半胱氨酸交联，作为 酶的磷酸化状态 3)金属会发生什么变化 结合位点在反应周期的P型ATP酶？ MgtB将是 使用6xHis标签纯化。 与J.K.博士合作布雷西， 纯化的洗涤剂增溶的酶将通过共价键连接在一起， 细胞质外半胱氨酸与有机半胱氨酸的巯基端基表面结合， 化学吸附在Ge/Si多层膜表面的自组装单层膜 衬底 这将提供载体定向的酶以确定 MgtB的轮廓结构使用非共振X射线衍射，从而 更牢固地建立其膜域结构。 在这 轮廓结构，利用共振x射线衍射，分布 结合的Ni 2+，作为酶的高亲和力位点上Mg 2+的替代物， 那就下定决心

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.