STRUCTURE, FUNCTION & FOLDING OF AN RNA BINDING PROTEIN

结构、功能

基本信息

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
项目类别：
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

项目摘要

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.

四螺旋束结构基序存在于多种蛋白质中执行广泛的功能。该提案的重点是确定该基序的结构和稳定性的基础，使用 Rop（罗姆）作为模型系统。 Rop 是两个螺旋-环-螺旋的同源二聚体单体，每个单体长63个氨基酸。 Rop 的函数是与特定的 RNA 复合物结合。由于这种蛋白质-RNA 相互作用，ColE1 质粒中的质粒拷贝数受到调节。 x- Rop 的射线晶体结构以 1.7A 分辨率已知，其 1H NMR 光谱已指定，尺寸小，便于分子分析造型。我们建议采用野生型Rop并通过系统重新设计和简化以确定必要的结构特征以获得稳定折叠的功能性蛋白质。三个类别的我们将研究的蛋白质是： 1) 内部重新包装的蛋白质。 Rop的内部包装可以解释为八次接收一个特征性的四个氨基酸“层”。这些层垂直于束的长轴。我们的目标是划定哪一层重新设计将产生一个正确且稳定的问题，并使我们能够以系统的方式重新包装整个蛋白质。 2) 连接回路的作用。连接环的作用指导蛋白质折叠或影响蛋白质稳定性都不是意愿明白了。我们将研究增加循环长度如何影响蛋白质稳定性和蛋白质折叠动力学受到以下影响循环长度和灵活性有系统地变化。 3）身份识别参与RNA识别的残基。 Rop为我们提供了难得的有机会了解已知小蛋白质对 RNA 的识别结构。我们将使用定点诱变来识别哪些残基参与RNA结合并决定其效果蛋白质-RNA复合物形成能量学上的特定突变。该数据将在结构信息的背景下进行解释来自我们的合作者 D. Crothers 教授的复合物，NMR 研究。将用于表征蛋白质的技术包括 CD、一维和二维 NMR 以及 X 射线晶体学结构特征； CD、荧光和量热法监测展开转变并确定蛋白质的能量学折叠式的;凝胶迁移分析和羟基自由基足迹法来确定蛋白质的能量学； RNA相互作用并比较细节不同的蛋白质-RNA复合物。