DETERMINATION OF 3 DIMENSIONAL STRUCTURES OF MACROMOLECULES IN SOLUTION BY NMR

该实验室的研究主要集中在利用多维核磁共振光谱对蛋白质、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，并开发和应用新的核磁共振和计算方法来辅助这些研究。特别强调的是参与信号转导和转录调节的复合体，以及艾滋病和与艾滋病有关的蛋白质。最近的成就包括将核磁共振方法的适用性扩展到大于40 kDa的结构，包括确定SIV gp41完整的44 kDa三聚体外结构域和细菌磷酸转移酶（PTS）系统的酶I和HPr之间的40 kDa磷酸化转移复合物的溶液结构。其他结构最近得到解决的系统包括：IIA酶（葡萄糖）和PTS途径HPr的磷酸化转移复合物，转录因子GATA-1、AREA、HMG-I/Y和MEF2A与DNA的复合物，野生型SRY（雄性性别决定因子）和SRY的性别反转突变体与DNA的复合物，抗hiv蛋白氰病毒素单体和结构域交换二聚体形式，负责保护逆转录病毒dna免受自整合的细胞因子BAF，以及HIV整合酶的N端和c端结构域。方法发展的例子包括在NIH开发的3D和4D异核磁共振实验的应用，这些实验对于研究较大的蛋白质至关重要，这些蛋白质的重叠共振构成了一个可怕的问题；利用排列张量的各向异性（例如，在溶解于稀释液晶介质中的大分子上测量的残余偶极耦合，例如，形病毒颗粒的向列相）或扩散张量（对于高度非球形分子）来提供远程定向信息的方法，这些信息无法从完全依赖于原子的紧密空间接近的其他核磁共振参数中获得；以及基于所有可用的实验核磁共振约束的快速有效的核磁共振光谱分析和三维结构计算算法的发展。最近的发展包括基于分子间NOE和偶极偶联数据，使用刚性调制最小化和约束/约束模拟退火来精确对接蛋白质-蛋白质复合物。

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.