PROTEIN NMR STRUCTURE AND DYNAMICS VIA ISOTOPIC LABELING

各种同位素标记技术正在开发的核磁共振 蛋白质和蛋白质-DNA复合物的结构和动力学分析。 大部分的努力都集中在建立选择性氘化 程序，使标记模式的各种个别残留物 类型可以几乎独立地同时在一个 实验性增长。 这就需要适当综合 氘代氨基酸以及构建合适的营养缺陷型 表达系统我们以前的工作表明，除了 预计光谱分辨率的增强，将有4-6倍 涉及碳结合的交叉分析灵敏度的提高 质子 这将显著增加聚合物的分子量范围。 大分子适合详细的结构分析。 其他潜在 有用的标记模式（例如交替的12 C-13 C富集）将 也被调查。 这些标记模式将用于确定溶液构象 真核生物转录因子。 虽然几个半胱氨酸锌 结合域已通过NMR分析， 需要更大的蛋白质结构域以包括指定DNA的区域 序列特异性结合。 对这一过程的深入了解将来自 148个残基HAP 1片段结合两个不同控制序列的研究 使用不同但重叠的结合位点。 在相关项目中，-100 Fos和Jun的残基结构域已经显示出选择性地结合到 AP-1 DNA结合位点为异二聚体。 由于DNA复合物为40 kD， 可以预期的是，所描述的选择性氘化方法将 这一模型系统的详细NMR结构分析是必要的， 癌基因功能 蛋白质动力学的研究将涉及酰胺捕获实验的E。杆菌 和T4硫氧还蛋白，以监测 折叠过程 的构象和动力学的平衡分析 一个模型“熔融球”蛋白质折叠中间状态将使 关键使用选择性氘化技术在这里开发的， 以克服严重的光谱分辨率问题。

Various isotopic labeling techniques are being developed for the NMR analysis of structure and dynamics of proteins and protein-DNA complexes. Much of the effort is focused on establishing selective deuteration procedures so that the labeling patterns of the various individual residue types can be, nearly independently, simultaneously controlled within a given experimental growth. This entails the appropriate synthesis of the deuterated amino acids as well as construction of a suitable auxotrophic expression system. Our previous work indicates that in addition to the anticipated enhancement in spectral resolution, there will be a 4-6 fold enhancement in the sensitivity of crosspeaks involving carbon bound protons. This will substantially increase the molecular weight range of macromolecules amenable to detailed structural analysis. Other potentially useful labeling patterns (such as an alternating 12C-13C enrichment) will also be investigated. These labeling patterns will be used to determine the solution conformation of eukaryotic transcription factors. Although several cysteine-zinc binding domains have been analyzed by NMR, structural determination of a larger protein domain is needed so as to include the regions specifying DNA sequence-specific binding. Insight into this process will come from the study of a 148 residue HAP1 fragment binds two dissimilar control sequences using distinct but overlapping binding sites. In a related project, -100 residue domains of Fos and Jun have been shown to bind selectively to the AP-1 DNA binding site as a heterodimer. As the DNA complex will be 40kD, it is anticipated that the selective deuteration approach described will be essential for detailed NMR structural analysis of this model system for oncogene function. Protein dynamics studies will involve amide trapping experiments on E. coli and T4 thioredoxins in order to monitor the detailed kinetics of the folding process. Equilibrium analysis of the conformation and dynamics of a model "molten globule" protein folding intermediate state will make critical use of the selective deuteration techniques developed here in order to overcome the severe spectral resolution problem.