AvanceIII 700MHz NMR spectrometer with cryogenic probe

描述（由申请人提供）：我们正在申请资金，以获得一台700 mhz溶液核磁共振光谱仪，以满足哈佛医学院（HMS）快速增长的高场核磁共振访问需求，用于具有挑战性的生物系统，特别是膜蛋白的结构研究。最近，Chou和Wagner实验室率先使用溶液核磁共振获得膜蛋白的原子分辨率结构，例如流感病毒的M2 H+通道结构（Schnell & Chou, Nature 2008）和线粒体的电压门控阴离子通道（VDAC-1） （Hiller等人，Science 2008）。这一成功刺激了一波涉及核磁共振膜蛋白结构表征的合作。正在进行的项目具有很高的学术和医学意义，但由于HMS的核磁共振设备老化，他们的进展缓慢。新的光谱仪将大大加快项目的进度，主要由James Chou （PI）和主要用户包括Gerhard Wagner， Ellis Reinherz, Christopher Walsh, Stephen Harrison， Bing Chen和Kai Wucherpfennig来操作和维护。下列项目将立即受益于该文书。(1)在继续关注质子易位子的过程中，Chou实验室将确定乙型流感病毒BM2 H+通道的结构，为设计新的抗流感化合物提供结构细节。它们还将完成线粒体内膜中32kda蛋白UCP2的结构，该蛋白解耦H+电化学电位以产生热量。UCP2抑制剂可以恢复胰腺2细胞的葡萄糖感知，用于治疗2型糖尿病。(2) Wagner实验室将确定VDAC-2与促凋亡蛋白Bak之间识别的结构基础。破坏VDAC-2/Bak与小分子的相互作用可能具有抗肿瘤活性。在另一个项目中，他们将研究参与RNA翻译起始的大分子复合物。(3) Reinherz小组将研究HIV-1 MPER结构域的跨膜片段连接到脂质小体的结构。结构细节将指导HIV疫苗的设计。(4)同样为了指导HIV疫苗的设计，Chen和Harrison实验室将通过核磁共振确定难以结晶的HIV gp41融合蛋白的融合中间态结构。(5) wucherpfenni实验室将继续研究免疫受体的膜区域，通过解决来自自然杀伤细胞的激活免疫受体复合物的结构，包括二聚体DAP12信号模块的跨膜结构域和NKG2C受体链。(6) Walsh实验室对肠obactin非核糖体肽合成酶的机制有着长期的兴趣，肠obactin非核糖体肽合成酶是一种由多个功能域组成的大型酶。在确定了37 kDa的EntF结构域后（Frueh et al., Nature 2008），他们将求解50 kDa的c结构域的结构，并表征其与EntF和EntB的相互作用。结构细节可能导致新的抗生素的设计。非核磁共振专家的用户将与James Chou和Gerhard Wagner合作进行实验。

DESCRIPTION (provided by applicant): We are requesting funding for acquiring a 700-MHz solution NMR spectrometer to satisfy the rapidly rising need of high-field NMR access at Harvard Medical School (HMS) for structural studies of challenging biological systems, in particular, membrane proteins. Recently, the Chou and Wagner labs spearheaded the effort to obtain atomic resolution structures of membrane proteins using solution NMR, e.g., the structures of M2 H+ channel from influenza virus (Schnell & Chou, Nature 2008) and the voltage-gated anion channel (VDAC-1) of mitochondrion (Hiller et al, Science 2008). The success has spurred a wave of collaborations that involves structural characterization of membrane proteins by NMR. The ongoing projects are of high academic and medical interests, but their progress is slow due to the aging NMR facility at HMS. The new spectrometer, which would significantly accelerate the projects, will be largely operated and maintained by James Chou (PI) and major users including Gerhard Wagner, Ellis Reinherz, Christopher Walsh, Stephen Harrison, Bing Chen, and Kai Wucherpfennig. The following projects would immediately benefit from the instrument. (1) In their continued focus on proton translocators, the Chou lab will determine the structure of the BM2 H+ channel of influenza B virus, for providing structural details for designing new anti-flu compounds. They will also complete the structure of the 32 kDa protein in the mitochondrial inner membrane, UCP2, which uncouples the H+ electrochemical potential for heat generation. UCP2 inhibitors could restore pancreatic 2-cell glucose sensing, for the treatment of type-2 diabetes. (2) The Wagner lab will determine the structural basis of recognition between VDAC-2 and the pro-apoptotic protein Bak. Disrupting the VDAC-2/Bak interaction with small molecules may have anti-tumor activity. In another project, they will study the macromolecular complexes involved in the RNA translation initiation. (3) The Reinherz group will study the structures of the HIV-1 MPER domain with transmembrane segment attached to the lipid bicelles. Structural details will guide HIV vaccine design. (4) Also in an effort to guide HIV vaccine design, the Chen and Harrison labs will determine by NMR the structure of the fusion intermediate state of the HIV gp41 fusion protein, which is difficult to crystallize. (5) The Wucherpfennig lab will continue their study of the membrane regions of immunoreceptors, by solving the structure of an activating immune receptor complex from the Natural Killer cells, consisting of the transmembrane domains of the dimeric DAP12 signaling module and the NKG2C receptor chain. (6) The Walsh lab has a long-term interest in the mechanism of the enterobactin non-ribosomal peptide synthetase, a mega enzyme consisting of multiple functional domains. After determining the structure of the 37 kDa EntF domain (Frueh et al., Nature 2008), they will solve the structure of the 50 kDa C-domain and characterize its interactions with EntF and EntB. Structural details may lead to the design of new antibiotics. Users who are not NMR experts will carry out the experiments in collaboration with James Chou and Gerhard Wagner. PUBLIC HEALTH RELEVANCE: Studies of the viral proton channels by the Chou lab will inspire new therapeutic approaches for fighting flu infections, whereas the HIV related projects from the labs of Reinherz, Harrison, and Chen will guide HIV vaccine design. The mitochondrial voltage-gated anion channel is one of the centerpieces in the programmed cell-death, and thus understanding its interaction with the pro- and anti-apoptotic factors can potentially identify new cancer therapeutic targets. The proposed 700 MHz NMR spectrometer would provide us sufficient instrumentation to move the above studies forward, while maintaining the postdoctoral fellow positions involved in these projects; it has no environmental impact.