PROTEIN NMR STRUCTURE AND DYNAMICS VIA ISOTOPIC LABELING

人们正在为核磁共振开发各种同位素标记技术 蛋白质和蛋白质-DNA复合体的结构和动力学分析。 大部分努力都集中在建立选择性重氢上。 程序使不同个体残留物的标记模式 类型几乎可以独立地同时在 在实验生长的情况下。这需要适当地合成 以及构建一种合适的营养缺陷型 表达系统。我们之前的工作表明，除了 预计光谱分辨率将提高4-6倍 提高涉及碳结合的相声的灵敏度 质子。这将大大增加其分子量范围。 易于进行详细结构分析的大分子。其他潜在的 有用的标记模式(例如交替的12C-13C浓缩)将 也被调查。 这些标记图案将用于确定溶液构象 真核转录因子。尽管几种半胱氨酸锌 结合结构域用核磁共振进行了分析，确定了其结构 需要更大的蛋白质结构域才能包括指定DNA的区域 序列特定的结合。对这一过程的洞察将来自 148个残基HAP1片段与两个不同控制序列结合的研究 使用不同但重叠的结合部位。在一个相关项目中，-100 Fos和Jun的残基结构域已经被证明选择性地结合到 AP-1DNA结合位点为异源二聚体。由于DNA复合体将是40kD， 预计所述的选择性氢化方法将是 对这个模型体系进行详细的核磁共振结构分析是必不可少的 癌基因功能。 蛋白质动力学研究将包括在大肠杆菌上进行的酰胺捕获实验 和T4硫氧还蛋白，以监测详细的动力学 折叠过程。分子的构象和动力学的平衡分析 一种“熔化的球状”蛋白质折叠中间态模型将使 在这里开发的选择性氢化技术的关键使用 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.