STRUCTURE, FUNCTION & FOLDING OF AN RNA BINDING PROTEIN

结构、功能

• 批准号: 2415189
• 负责人: LYNNE J. REGAN
• 金额: $ 16.81万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415189
• 关键词: RNA; RNA binding protein; X ray crystallography; calorimetry; chemical kinetics; chemical models; chemical stability; circular dichroism; conformation; crystallization; fluorescence spectrometry; gel filtration chromatography; gel mobility shift assay; intermolecular interaction; nuclear magnetic resonance spectroscopy; physical model; protein denaturation; protein engineering; protein folding; protein sequence; protein structure; protein structure function; site directed mutagenesis; structural biology; thermodynamics

The four-helix bundle structural motifs is found in a variety of proteins that perform a wide range of functions. This proposal focuses upon determining the basis of the structure and stability of this motif, using Rop (Rom) as a model system. Rop is a homo-dimer of two helix-loop-helix monomers, each of which is 63 amino acids long. The function of Rop is to bind to a specific RNA complex. As a consequence of this protein-RNA interaction, plasmid copy number in ColE1 plasmids is regulated. The x- ray crystal structure of Rop is known at 1.7A resolution, its 1H NMR spectrum has been assigned and its small size is convenient for molecular modelling. We propose to take wild-type Rop and by systematic re-design and simplication to determine the structural features that are essential for a stably folded and functional protein. The three classes of proteins that we will study are: 1) Proteins with re-packed interiors. Rop's internal packing can be interpreted as eight receptions of a characteristic four amino acid "layer". The layers are perpendicular to the long axis of the bundle. Our aim is to delineate which layer redesigns will generate a correctly and stably problem and will allow us to approach the repacking of the entire protein in a systematic fashion. 2) Role of the connecting loops. The role connecting loops play in directing protein folding or i influencing protein stability is not will understood. We will investigate how increasing loop length affects protein stability and the kinetics of protein folding are affected as loop length and flexibility are systematically varied. 3) Identification of the residues involved in RNA recognition. Rop provides us with a rare opportunity to understand RNA recognition by a small protein of known structure. We will use site-directed mutagenesis to identify which residues are involved in RNA binding and will determine the effect of specific mutations on the energetics of protein-RNA complex formation. This data will be interpreted in the context of structural information on the complex that comes from our collaborator, Prof. D. Crothers, NMR studies. The techniques that will be used to characterize the proteins include CD, 1- and 2-dimensional NMR and x-ray crystallography for structural characterizations; CD, Fluorescence and Calorimetry to monitor the unfolding transition and to determine the energetics of protein folding; gel-shift assays and hydroxy-radical footprinting to determine the energetics of protein; RNA interactions and to compare the details of different protein-RNA complexes.

在多种蛋白质中发现了四螺旋束结构基序 该功能范围很广。 该提议的重点是 使用该基序的结构和稳定性的基础 ROP（ROM）作为模型系统。 ROP是两个Helix-Loop-helix的同型二聚体 单体，每个小体长63个氨基酸。 ROP的功能是 结合特定的RNA复合物。 由于该蛋白-RNA的结果 调节Cole1质粒中的相互作用，质粒拷贝数。 X- ROP的射线晶体结构以1.7A分辨率为1H NMR已知 频谱已被分配，其小尺寸方便于分子 造型。 我们建议采用野生型ROP，并通过系统的重新设计 并简化以确定必不可少的结构特征 用于稳定折叠和功能性蛋白质。 这三个类 我们将研究的蛋白质是：1）带有重新包装内部的蛋白质。 ROP的内部包装可以解释为 特征四个氨基酸“层”。 这些层垂直于 捆绑包的长轴。 我们的目的是描述哪一层 重新设计将产生正确且稳定的问题，并将允许我们 以系统的方式处理整个蛋白质的重新包装。 2）连接循环的角色。 连接循环的角色在 指导蛋白质折叠或我影响蛋白质稳定性不是 理解。 我们将研究循环长度的增加如何影响 蛋白质稳定性和蛋白质折叠的动力学受到影响 循环长度和灵活性是系统的。 3）识别 涉及RNA识别的残基。 ROP为我们提供了罕见的 通过已知的小蛋白质了解RNA识别的机会 结构。 我们将使用以网站为导向的诱变来确定哪个 残基参与RNA结合，将确定 关于蛋白-RNA复合物形成能量学的特异性突变。 这些数据将在结构信息的背景下进行解释 关于我们的合作者D. Crothers教授，NMR的综合大楼 研究。 将用于表征蛋白质的技术 包括CD，1和2维的NMR和X射线晶体学用于 结构特征； CD，荧光和量热法以监测 展开的过渡并确定蛋白质的能量学 折叠式的;凝胶转移测定和羟基激进足迹以确定 蛋白质的能量； RNA相互作用并比较细节 不同的蛋白-RNA复合物。