NMR STUDIES OF PROTEINS AND PROTEIN-DNA BINDING

该计划的目的是了解特定的蛋白质-DNA 溶液中的识别和结合动力学 NMR光谱和 基因工程方法正被用于表征结构和 互动的功能特点。 下的生物系统 研究是甲硫氨酸脱辅阻遏蛋白二聚体，metJ，其 辅阻遏物S-腺苷甲硫氨酸，以及该蛋氨酸的合成片段 操纵基因 根据最近的晶体学数据，这种抑制子 蛋白质似乎代表了一个新的DNA结合基序，涉及插入 两个反平行链，一个来自每个蛋白质单体，进入 主要凹槽的双工，并提供了一个复杂的，但易于处理的系统 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 在蛋氨酸调节子中。 为了实现这些目标，异位和同位NMR方法 正与光谱编辑技术一起沿着用于蛋白质 用C-13和N-15同位素均匀标记。 使用特定的共振 在蛋白质和不同的操纵子片段中的分配 共有序列DNA的长度和位置， 对含有和不含S-腺苷甲硫氨酸的结合复合物进行了探测 通过1D光谱监测的蛋白质-DNA滴定研究， 1D和2D核Overhauser效应光谱。 光谱的使用 用标记的蛋白质结合到未标记的DNA的编辑技术， SAM计划探索结合蛋白质、DNA和 SAM溶液。 所选突变体的DNA结合特性 还将研究含有单个氨基酸取代的蛋白质 基于凝胶移位和DNA酶保护筛选的结果。 与 晶体学表示作为出发点，动力学特征 将在溶液中系统地检查这种NEV结合基序。

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.