CHARACTERIZATION OF MEMBRANE PROTEIN BY SSNMR

描述：（申请人摘要的逐字逐句）我们建议进行 质子泵的结构研究，F0从ATP合酶，涉及高 均匀13 C，15 N标记的C的场固态NMR距离测量 在这个复合体中。许多方法已经过验证， 水合的微晶蛋白质，如BPTI和泛素。的 这种复合物的机械关键c亚基将是一个理想的试验案例 用于膜蛋白的分配，因为它只有79个残基， 可能有许多分散良好的NMR谱线。魔角旋转， 各向同性碳化学位移的高分辨率测量，类似于 溶液NMR将用于确认侧链身份和二级 结构基于有效的C-C和C-N转换的3D脉冲序列将被 用于建立亚基的位点特异性主链和侧链分配 C.我们最近证明了高分辨率多维固态 NMR方法可用于关联化合物的主链和侧链化学位移。 水合微晶U-13 C，15 N BPTI和固有膜 蛋白质，LH 1。典型的13 C峰线宽为0.5 ppm;位移之间的一致性 对于固态和溶液中的BPTI测量，通常优于0.5 ppm. N-C和C-C传输都可以高效率地执行，因此， 允许通过主干的顺序分配。将探测距离 通过对指定侧链碳的多维研究，他们还将 通过空间距离，用选择性闪烁和19 F-19 F探测 测量.一致标记的亚基c和合酶复合物是可用的 通过与马克吉尔文教授（爱因斯坦学院）的合作， 医学）。一些标记的样品已经制备了许多mg 规模和仪器时间高场固体机器将提供 一年亚基c将在完整的F0复合体中进行研究; 尽管该复合体相当大（ca. 200 kDa），我们仍然可以期待非常 与c亚基相关的强信号，因为在c亚基中有12个拷贝。 复合物相比，只有1-2个拷贝的所有其他肽，和亚基c 约为F0质量的一半。亚单位c也将在 重组脂质包埋的C12寡聚体的背景，使得混合同位素 标记策略可用于区分分子内和 分子间接触我们将测试现有结构的相关性 低聚物的模型，特别是假设Asp 61的电离， 质子泵的核心参与者，引发了机械上重要的全球 ATP合成酶的构象变化。

Description: (Verbatim from the applicant's abstract) We propose to carry out structural studies of the proton pump, F0 from ATP synthase, involving high field solid state NMR distance measurements of the uniformly 13C, 15N labeled c subunit in this complex. Many of the methods have been validated using hydrated, micro-crystalline proteins such as BPTI and ubiquitin. The mechanistically crucial c subunit of this complex will be an ideal trial case for assignments in membrane proteins, since it has only 79 residues, and probably has many well dispersed NMR lines. Magic angle spinning, high-resolution measurements of isotropic carbon chemical shifts, analogous to solution NMR, will be used to confirm sidechain identity and secondary structure. 3D pulse sequences based on efficient C-C and C-N transfers will be used to establish site-specific backbone and sidechain assignments for subunit c. We recently demonstrated that high-resolution multidimensional solid state NMR methods can be used to correlate backbone and side-chain chemical shifts of hydrated micro-crystalline U-13C, 15N BPTI and of the intrinsic membrane protein, LH1. Typical 13C peak linewidths are 0.5 ppm; agreement between shifts measured for BPTI in the solid state and solution is typically better than 0.5 ppm. Both N-C and C-C transfers can be performed with high efficiency, thus allowing sequential assignments through the backbone. Distances will be probed with multidimensional studies of the assigned sidechain carbons; they will also be probed with selective fluorination and 19F-19F through space distance measurements. Uniformly labeled subunit c and synthase complex are available through collaboration with Professor Mark Girvin (Einstein College of Medicine). Some of the labeled samples have already been prepared at many mg scale, and instrument time on high field solids machines will be available in one year. Subunit c will be studied in the context of an intact F0 complex; although this complex is quite large (ca. 200 kDa) we can still expect very strong signals associated with the c subunit since there are 12 copies in the complex as compared with only 1-2 copies of all other peptides, and subunit c represents about half of the mass of F0. Subunit c will also be studied in the context of reconstituted lipid embedded c12 oligomers, so that mixed isotopic labeling strategies can be used to distinguish intramolecular and intermolecular contacts. We will test the relevance of existing structural models for the oligomer, particularly the hypothesis that ionization of Asp61, the central player in proton pumping, provokes mechanistically important global conformational changes in ATP synthase.