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有了很大发展。这些设施的重点一直是结构和机械生物学。这些新设施包括：a)一套可容纳三名教职员工和受训人员的X射线结晶学设备；b)一套拥有三台仪器的质谱学设备；c)一套用于微尺度多肽合成、分离、测序和分析的设备；d)一个用于时间分辨分子光谱学的激光光谱分析实验室，可为广大教职员工和受训人员提供支持；e)布鲁克海文国家实验室的一条同步加速器光束线，目前支持多个系教职员工的研究和培训活动。这些新设备增加了脉冲EPR波谱(Jack Peisach博士)和生物核磁共振(Raj Gupta博士)现有计划的优势。总之，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主要培训地点之一。每年，在大约300名博士后和博士后学员中，有更大一部分人参与了结构生物学培训，作为他们研究计划的一部分。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，这种设备是用当前技术确定生物分子结构所需的类型。这一不足之处在过去两次实地考察该校结构生物学和生物物理学培训补助金的书面批评中都有提及。本提案描述了一项通过建设结构核磁共振资源来弥补这一弱点的计划，其中包括两台光谱仪和相关的用于分子建模的工作站：600 MHz 1H13C15N三共振2.5和5 mm探头、4个通道系统、3轴梯度300 MHz 1H13C31P19F 5 mm探头和10 mm宽带多核探头、2个通道4个175 MHz UNIX图形工作站，位于设施内用于离线数据分析的设施内。一位非常合格的教职员工Mark Girvin博士已被聘请来监督该设施。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，供所有教职员工及其受训人员使用。教员及其受训人员正在调查的研究问题的例子包括：来自大肠杆菌的F1Fo ATP合成酶跨膜亚单位c的结构，茎环RNA二级结构的融化速度，以及RNA结合蛋白结构/功能关系对构成Chromoranin A1-40蛋白定位结合位点结构的结构域的细胞表面N-连接复杂碳水化合物结构的影响，一种二硫键分泌蛋白和膜蛋白连接区的AA-取代结构P-糖蛋白用15N标记的酪氨酸磷酸酶的核磁共振法测定催化部位的运动用NOE分析碳水化合物与凝集素的结合和顺磁效应除了促进这些研究项目外，结构核磁共振资源还将被用作通过核磁共振解决结构问题的理论和实践方面的实践高级研究生院课程的站点。这门课程每两年教授一次，所以所有的博士生都有机会学习结构分析的核磁共振方法。结构核磁共振资源中图形工作站的位置将有助于使用实验室进行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.