NMR STUDIES OF PROTEINS AND PROTEIN-DNA BINDING

描述：本计划的总体目标仍然是了解 特异性蛋白质-DNA识别和结合动力学。 NMR 光谱学和基因工程方法被用来表征 这种相互作用的结构、功能和动态特征。 的 研究中的生物系统是甲硫氨酸脱辅基阻遏蛋白 二聚体、metJ、其辅阻遏物S-腺苷甲硫氨酸和合成片段 有一个指纹 根据最近的晶体学数据， 阻遏蛋白代表了一种新的DNA结合基序， 两个反平行链，一个来自每个蛋白质单体，进入主要的 双链体DNA的沟，并提供了一个完整的和易于处理的系统， 详细的NMR研究。 总体努力旨在产生一个 更好地了解这些相互作用如何控制表达 蛋氨酸调节子中的基因。 为了实现这些目标，异质性和homeland，多维 NMR方法正在开发中，并与光谱编辑一起沿着使用 研究蛋白质及其溶液动力学和结构的技术 配合物 辅阻遏物增强DNA结合的机制 抑制是特别感兴趣的，因为它似乎影响绑定 从晶体结构中长距离观察。 溶剂检查方法 通过快速质子交换暴露在蛋白质和蛋白质-DNA复合物中， 正在开发，以提供对系统动态行为的洞察力 在溶液中。 蛋白质骨架动力学也在免费的 和结合的蛋白质。 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， 根据凝胶迁移和DNA酶保护筛选的结果， 通过NMR方法研究。

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.