NMR STUDIES OF PROTEINS AND PROTEIN-DNA BINDING

描述：本计划的总体目标尚待了解 溶液中特异的蛋白质-DNA识别和结合动力学。核磁共振 光谱学和基因工程方法正被用来表征 这种相互作用的结构、功能和动态特征。这个 正在研究的生物系统是蛋氨酸抑制蛋白 二聚体、MetJ及其辅阻遏子S-腺苷蛋氨酸及其合成片段 大气局操作者的DNA。根据最新的结晶学数据，这 阻遏蛋白是一种新的DNA插入结合基序 两条反平行的链，每个蛋白质单体一条，进入主要的 双链DNA的凹槽，并提供了一个完整和易处理的系统 详细的核磁共振研究。总体努力旨在产生一个 更好地理解这些相互作用如何控制 蛋氨酸调节子中的基因。 为了实现这些目标，异核和同核，多维 核磁共振方法正在开发和使用，并与光谱编辑一起使用 研究蛋白质及其结构的溶液动力学和结构的技术 复合体。辅阻遏子增强DNA结合和 压抑特别有意义，因为它似乎会影响约束 从晶体结构中的很远距离。检测溶剂的方法 通过快速质子交换暴露在蛋白质和蛋白质-DNA复合体中 被开发为提供对系统的动态行为的洞察 在溶液中。蛋白质骨架动力学也在探索中。 和结合蛋白的异核磁化松弛方法。 用稳定同位素C-13、N-15和H-2标记的MetJ正在用于 这些研究。所选突变蛋白的DNA结合特性， 根据凝胶漂移和DNA酶保护筛选的结果，还将 用核磁共振方法进行了研究。

DESCRIPTION: The overall objective of this program remains to understand specific protein-DNA recognition and binding dynamics in solution. NMR spectroscopy and genetic engineering methods are being used to characterize structural, functional, and dynamic features of this interaction. The biological system under investigation is the methionine aporepressor protein dimer, metJ, its corepressor S-adenosylmethionine, and synthetic fragments of the met operator DNA. Based on recent crystallographic data, this repressor protein represents a novel DNA binding motif involving insertion of two antiparallel strands, one from each protein monomer, into the major groove of the duplex DNA and provides a complete and tractable system for detailed NMR investigation. The overall effort is designed to generate a better understanding of how these interactions control the expression of genes in the methionine regulon. To achieve these objectives, heteronuclear and homonuclear, multidimensional NMR methods are being developed and used along with spectral editing techniques to study solution dynamics and structures of the protein and its complexes. The mechanism by which the corepressor enhances DNA binding and repression is of particular interest, since it appears to influence binding from long distances in the crystal structure. Methods to examine solvent exposure in the protein and protein-DNA complex via fast proton exchange are being developed to provide insight into the dynamic behavior of the system in solution. Protein backbone dynamics are also being explored in the free and bound protein using heteronuclear magnetization relaxation methods. MetJ labeled with the stable isotopes C-13, N-15, and H-2 is being used in these studies. The DNA binding characteristics of mutant proteins selected, based on results of gel shift and Dnase protection screening, will also be studied by NMR methods.