Acquisition of a Structural NMR Resource

阿尔伯特·爱因斯坦医学院的共享培训和研究设施在过去十年中得到了很大发展。这些设施的重点是结构和机械生物学。这些新设施包括a）一个x射线晶体学套件，配有三名教职员工和受训人员的设施，B）一个质谱仪设施，配有三台仪器，教职员工，c）一个微尺度的设施， 肽 合成，分离， 测序 d）一个用于时间分辨分子光谱学的激光光谱学实验室，支持广泛的教师和学员，e）布鲁克海文国家实验室的同步加速器光束线，目前支持几个部门的教师的研究和培训活动。 这些新设施增加了脉冲EPR光谱（Jack Peisach博士）和生物NMR（Raj Gupta博士）现有项目的优势。 这些设施使阿尔伯特·爱因斯坦医学院成为大纽约地区结构生物学的首要培训场所之一。每年，在大约300名博士中，和博士后学员参与结构生物学培训作为他们的研究计划的一部分。 我们的培训和研究计划中的一个弱点是缺乏多共振高场核磁共振设备的类型所需的结构测定的生物分子由目前的技术。这一缺陷在过去两次实地考察的书面批评中提到，在这所学校的结构生物学和生物物理学培训赠款。 该提案描述了一项计划，通过构建包含两个光谱仪和相关分子建模工作站的结构NMR资源来弥补这一弱点： 600 MHz 1H 13 C15 N三重谐振2.5和5 mm探头，4 通道系统，3轴梯度 300 MHz 1H 13 C31 P19 F 5 mm探头和10 mm宽带 多核探针，2通道 4个175 MHz UNIX图形工作站，位于设施内，用于离线数据分析 该设施将新建，用于容纳该设备，并将位于Ullman研究大楼的底层，挖掘到地基基岩中。一位高素质的教职员工，马克吉尔文博士，已被招募来监督该设施。还将提供一名全职工作人员。结构核磁共振资源将作为一个共享的研究和培训设施提供给所有教师和他们的学员。 由教师和他们的学员调查的研究问题的例子包括： F1 Fo ATP跨膜亚基c的结构 合成酶。杆菌 茎环RNA二级结构的解链速率和 RNA结合蛋白的影响 细胞表面N-连接复合糖结构/功能关系 构成蛋白质定位结合位点的结构域的结构 二硫键分泌蛋白嗜铬粒蛋白A1-40的结构和AA-取代的影响 膜蛋白P-糖蛋白连接区的结构 用核磁共振法测定15 N标记酪氨酸磷酸酶的催化位点皮瓣运动 用NOE和顺磁效应分析糖与凝集素的结合 除了促进这些研究项目，结构NMR资源将被用作实践高级研究生院课程的网站，该课程涉及通过NMR解决结构问题的理论和实践。这门课每两年开一次，所以所有的博士生都要参加。学生将有机会学习核磁共振的结构分析方法。在结构核磁共振资源的图形工作站的位置将有利于使用实验室的研究生和研究生培训。

Shared training and research facilities at the Albert Einstein College of Medicine have been developed substantially during the past decade. The focus of these facilities has been in structural and mechanistic biology. These new facilities include a) an x-ray crystallography suite with facilities for three faculty members and trainees, b) a mass spectrometry facility with three instruments, faculty and staff, c) a facility for micro-scale peptide synthesis, isolation, sequencing and analysis, d) a laser spectroscopy laboratory for time-resolved molecular spectroscopy which supports a broad group of faculty and trainees, e) a synchrotron beam-line at Brookhaven National Laboratories which currently supports research and training activities of faculty members in several departments. These new facilities have added to the strengths of existing programs in pulsed EPR spectroscopy (Dr. Jack Peisach), and biological NMR (Dr. Raj Gupta). Together, these facilities make the Albert Einstein College of Medicine one of the premier training sites for structural biology in the greater New York area. Each year, a larger fraction of the approximately 300 Ph.D. and postdoctoral trainees are involved in structural biology training as part of their research programs. A weakness in our training and research programs has been the deficiency in multipleresonance high-field NMR equipment of the type required for structural determination of biological molecules by current technologies. This deficiency was mentioned in the written critiques for each of the past two site-visits for training grants in structural biology and biophysics at this school. This proposal describes a plan to remedy this weakness by the construction of a structural NMR resource containing two spectrometers and associated workstations for molecular modeling: 600 MHz 1H13C15N triple resonance 2.5 and 5 mm probes, 4 channel system, 3-axis gradients 300 MHz 1H13C31P19F 5 mm probe and 10 mm broadband multinuclear probe, 2 channels four 175 MHz UNIX graphics workstations located in the facility for offline data analysis The facility will be newly constructed for the purpose of housing this equipment and will be located on the ground floor of the Ullman Research Building, excavated into foundation bedrock. A highly qualified faculty member, Dr. Mark Girvin, has been recruited to oversee the facility. A full-time staff person will also be provided. The structural NMR resource will operate as a shared research and training facility available to all faculty and their trainees. Examples of research problems under investigation by faculty and their trainees include: structure of the transmembrane subunit c of F1Fo ATP synthase from E. coli melting rates of stem-loop RNA secondary structure and the influence of RNA binding proteins structure/function relationships for cell-surface N-linked complex carbohydrates structure of domains which constitute protein-localization binding sites structure of chromogranin A1-40, a disulfide secretory protein and the effect of AA-replacements structure of the linker region of the membrane protein P-glycoprotein determination of catalytic site flap movements by NMR of 15N-labeled tyrosine phosphatase analysis of the binding of carbohydrates to lectins by NOE and paramagnetic effects In addition to fostering these research programs, the structural NMR resource will be used as the site for a hands-on advanced level graduate school course in the theory and practice of solving structural problems by NMR. The course is to be taught every two years so all Ph.D. students will have the opportunity to learn NMR methods of structural analysis. The location of the graphics workstations in the structural NMR resource will facilitate use of the laboratory for graduate an d postgraduate training.