Protein Structure Determinations by NMR and Joint NMR-X-ray Refinement

9406065霍夫曼拟议研究的目标是分析两种不寻常的和生物学上重要的蛋白质分子的结构。待研究的具体结构是：（1）噬菌体T7的“0.3”蛋白;（2）原核核糖体的RNA结合蛋白L9。 在每种情况下，获得的结构信息将提供洞察蛋白质在执行其特定功能时所使用的机制。噬菌体T7的12.5kDalton 03基因产物具有独特的结合E. coli限制性内切酶和RNA聚合酶。该蛋白质执行抑制E. coli限制性酶切系统，从而允许T7噬菌体将其基因组安全地注入宿主细胞。有证据表明，0.3蛋白可能通过模仿通常被大肠杆菌识别的DNA结构来发挥功能。coli限制性内切酶系统。0.3蛋白质的结构将通过NMR方法确定，从而深入了解其结合和抑制DNA加工酶的机制。L9是原核生物核糖体中高度保守的一级RNA结合蛋白。在拟议的研究计划中，NMR和X射线数据将用于提供结构约束，蛋白质结构将通过对NMR和X射线数据的同时细化或“联合细化”来确定。这种联合细化方法应该产生更高分辨率的结构，其中两个RNA结合位点都被很好地定义。这种分析将提供一个模型，如何NMR和X射线数据可以实际和逻辑地结合起来，以确定一个单一的蛋白质分子的结构。 这项研究的主要目标是分析两种不寻常的、具有生物学意义的蛋白质分子的结构。待研究的具体结构是：1）一种蛋白质，其独特的结合和抑制DNA加工酶的功能的能力;和2）一种蛋白质，其具有识别和结合核糖体RNA的特定序列的能力。在每种情况下，获得的结构信息将提供洞察蛋白质在执行其特定功能时所使用的机制。拟议项目的另一个目标是利用核磁共振（NMR）实验和X射线衍射实验的综合数据开发确定蛋白质结构的新方法。预计这些新方法将有助于更准确地确定蛋白质结构。 ***

9406065 Hoffman The goal of the proposed research is to analyze the structures of two unusual and biologically significant protein molecules. The specific structures to be investigated are: (1) the "0.3" protein of bacteriophage T7; (2) the RNA-binding protein L9 from the prokaryotic ribosome. In each case, the structural information obtained will provide insight into the mechanism used by the protein in performing its particular function. The 12.5 kDalton 03 gene product of bacteriophage T7 is unique in its ability to bind to E. coli restriction enzymes and RNA polymerase. The protein performs the task of inhibiting the E. coli restriction system, thus permitting the T7 phage to safely inject its genome into the host cells. There is evidence suggesting that the 0.3 protein may function by mimicking the DNA structures that are normally recognized by the E. coli restriction system. The structure of the 0.3 protein will be determined by NMR methods, providing insight into the mechanism by which it binds and inhibits DNA-processing enzymes. L9 is a highly conserved primary RNA-binding protein from the prokaryotic ribosome. In the proposed research plan, NMR and x-ray data will be used to supply structural constraints, and the protein structure will be determined by a simultaneous refinement against both the NMR and x-ray data, or "joint-refinement". This joint-refinement approach should yield a much higher resolution structure, in which both RNA-binding sites are well defined. This analysis will provide a model as to how NMR and x-ray data can be practically and logically combined to determine the structure of a single protein molecule. %%% The principal goal of the proposed research is to analyze the structures of two unusual and biologically significant protein molecules. The specific structures to be investigated are: 1) a protein that is unique in its ability to bind to and inhibit the function of DNA-processing enzymes; and 2) a protein with the ability to recognize and bind specific sequences of ribosomal RNA. In each case, the structural information obtained will provide insight into the mechanism used by the protein in performing its particular function. An additional goal of the proposed project is to develop new methods for determining protein structures, using the combined data from nuclear magnetic resonance (NMR) experiments and x-ray diffraction experiments. It is anticipated that these new methods will be useful in determining protein structures with greater accuracy. ***