DETERMINATION OF 3 DIMENSIONAL STRUCTURES OF MACROMOLECULES IN SOLUTION BY NMR

核磁共振法测定溶液中大分子的三维结构

• 批准号: 6432092
• 负责人: G. MARIUS CLORE
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432092
• 关键词: DNA; DNA binding protein; Streptococcus; bacterial proteins; human immunodeficiency virus; integrase; ligands; method development; nuclear magnetic resonance spectroscopy; protein structure; solutions; structural biology; thioredoxin; transcription factor; transposon /insertion element; virus protein

Research in this laboratory is centered around solution studies on the structure and dynamics of proteins, protein-protein complexes and protein-nucleic acid complexes using multidimensional NMR spectroscopy, and on the development and application of novel NMR and computational methods to aid in these studies. Particular emphasis is being placed on complexes involved in signal transduction and transcriptional regulation, and on AIDS and AIDS-related proteins. Recent accomplishments include the extension of the applicability of the NMR method to structures larger than 40 kDa, including the determination of the solution structures of the complete 44 kDa trimeric ectodomain of SIV gp41 and the 40 kDa phosphoryl transfer complex between enyzme I and HPr of the bacterial phosphotransferase (PTS)system. Other systems whose structures have recently been solved include the phosphoryl transfer complex of enzyme IIA(glucose)and HPr of the PTS pathway, complexes of the transcription factors GATA-1, AREA, HMG-I/Y and MEF2A with DNA, complexes of wild-type SRY (the male sex determining factor) and a sex reversal mutant of SRY with DNA, the anti-HIV protein cyanovirinin monomeric and domain-swapped dimeric forms, the cellular factor BAF which is responsible for protecting retroviral DNAfrom autointegration, and the N- and C-terminal domains of HIV integrase. Examples of methodological developments include thep anoply of 3D and 4D heteronuclear NMR experiments that have been developed at the NIH and are essential for studying larger proteins whose overlapping resonances pose a formidable problem; methods that make use of anisotropy of the alignment tensor (e.g. residual dipolar couplings measured on macromolecules dissolved in dilute liquid crystalline media such as the nematic phases ofrod-shaped virus particles) or the diffusion tensor (for highly non-spherical molecules) to provide long-range orientational information that is not available from other NMR parameters that rely entirely on close spatial proximity of atoms; and the development of fast and efficient algorithms for the analysis of NMR spectra and for the computation of three-dimensional structures based on all available experimental NMR restraints. Very recent developments include the use of rigid modyminimization and constrained/restrained simulated annealing to accurately dock protein-protein complexes on the basis of intermolecular NOE and dipolar coupling data.

该实验室的研究主要集中在使用多维NMR光谱对蛋白质，蛋白质-蛋白质复合物和蛋白质-核酸复合物的结构和动力学进行溶液研究，以及开发和应用新型NMR和计算方法来帮助这些研究。特别强调的是参与信号转导和转录调节的复合物，以及艾滋病和艾滋病相关蛋白。最近的成就包括扩展的NMR方法的适用性大于40 kDa的结构，包括完整的44 kDa的SIV gp 41和40 kDa的磷酸转移酶I和细菌磷酸转移酶（PTS）系统的HPr之间的酶I和磷酸转移复合物的三聚体胞外域的溶液结构的测定。其结构最近已被解决的其它系统包括酶IIA（葡萄糖）和PTS途径的HPr的磷酰基转移复合物，转录因子加塔-1、AREA、HMG-1/Y和MEF 2A与DNA的复合物，野生型SRY的复合物（男性性别决定因子）和SRY的性别逆转突变体与DNA，抗艾滋病毒蛋白cyanovirinin单体和结构域交换的二聚体形式，负责保护逆转录病毒DNA免于自整合的细胞因子BAF，以及HIV整合酶的N-和C-末端结构域。方法学发展的例子包括3D和4D异谱NMR实验的应用，这些实验是NIH开发的，对于研究较大的蛋白质是必不可少的，这些蛋白质的重叠共振构成了一个可怕的问题;利用排列张量各向异性的方法（例如，对溶解在稀释液晶介质中的大分子（如杆状病毒颗粒的双相）测量的残余偶极偶联）或扩散张量（对于高度非球形分子），以提供从完全依赖于原子的紧密空间接近的其他NMR参数无法获得的长程取向信息;以及开发用于NMR光谱分析和基于所有可用实验NMR约束计算三维结构的快速有效算法。最近的发展包括使用刚性modymimminization和约束/约束模拟退火，以准确对接蛋白质-蛋白质复合物的基础上的分子间NOE和偶极耦合数据。