NMR STUDIES OF PROTEINS AND PROTEIN-DNA BINDING

这个项目的目标是了解特定的蛋白质-DNA 溶液中的识别和结合动力学。核磁共振波谱和 基因工程方法正被用来表征结构和 互动的功能特征。中国的生物系统 研究的是蛋氨酸抑制蛋白二聚体，MetJ，ITS 辅抑制子S-腺苷蛋氨酸和MET的合成片段 操作者DNA。根据最新的结晶学数据，这种抑制物 蛋白质似乎代表了一种新的DNA结合基序，涉及插入 两条反平行的链，每个蛋白质单体一条，进入 双工的主要凹槽，并提供了一个复杂但易处理的系统 进行详细的核磁共振研究。总体目标是更好地 了解这些相互作用如何控制基因的表达 在蛋氨酸调节剂中。 为了实现这些目标，异核和同核核磁共振方法 与蛋白质的光谱编辑技术一起使用 统一标记C-13和N-15同位素。使用特定的共振 在蛋白质和变种的操纵子片段中的指认 共有序列DNA的长度和位置，接触区在 结合和不结合S-腺苷蛋氨酸的络合物正在探索中 通过一维光谱监测的蛋白质-DNA滴定研究 一维和二维核Overhaser效应谱。光谱的使用 用标记的蛋白质结合到未标记的DNA和 SAM计划探索结合蛋白、DNA和 萨姆在解决问题。选育突变体的DNA结合特性 含有单一氨基酸取代的蛋白质也将被研究。 根据凝胶漂移和DNA酶保护筛选的结果。与 以晶体表象为起点，动力学特征 将在溶液中系统地研究这个NEV结合基序的。

The objective of this program is to understand specific protein-DNA recognition and binding dynamics in solution. NMR spectroscopy and genetic engineering methods are being used to characterize structural and functional features of the 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 appears to represent a new DNA binding motif involving insertion of two antiparallel strands, one from each protein monomer, into the major groove of the duplex, and provides a complex but tractable system for detailed NMR investigation. The overall goal is a better understanding of how these interactions control the expression of genes in the methionine regulon. To achieve these objectives, heteronuclear and homonuclear NMR methods are being used along with spectral editing techniques on protein uniformly labeled with C-13 and N-15 isotopes. Using specific resonance assignments in both the protein and in operator fragments of varying length and position of the consensus sequence DNA, the contact regions in the bound complex with and without S-adenosylmethionine are being probed via protein-DNA titration studies monitored by 1D spectroscopy and both 1D and 2D nuclear Overhauser effect spectroscopy. Use of spectral editing techniques with the labeled protein bound to unlabeled DNA and SAM is planned to explore configurations of the bound protein, DNA, and SAM in solution. The DNA binding characteristics of selected mutant proteins containing single amino acid substitutions will also be studied based on results of gel shift and DNase protection screening. With the crystallographic representation as a starting point, dynamical features of this nev binding motif will be systematically examined in solution.