Structural Studies of Spectrin

血影蛋白的结构研究

• 批准号: 9407779
• 负责人: Leslie Wo-Mei Fung
• 金额: $ 30万
• 依托单位: Loyola University of Chicago
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9407779&HistoricalAwards=false
• 关键词: Structural; Studies; Spectrin

9407779 Fung We propose to understand the structural of the human erythrocyte spectrin -subunit in forming head-to-head association with the -subunit to give heterodimers and tetramers. We will use cDNA and PCR methods to express specific -subunit segments in E. coli, with a glutathione-S-transferase gene fusion system. Spectrin segments selected for this study will start from the N-terminus of the - subunit and ending at different positions in the sequence, giving peptides of various lengths. Binding studies of these purified, -recombinant peptides with the isolated -subunit will be conducted to select peptides with functional properties. From the functional peptides, Fourier transform infrared techniques will be used to select the peptides that have secondary structure similar to that of intact spectrin. The shortest peptide of these peptides will be subjected to site directed mutagenesis to provide a set of peptides, each with one cysteine per peptide but at different positions in the sequence, to obtain topological information by spin label electron paramagnetic resonance methods. At the conclusion of this project, we expect to have obtained structural information about the first 60 amino acids in the N-terminal region of the -submit that are required for head-to-head association. We believe that an understanding of the -and -subunit head-to-head association forms the basis for further understanding the assembly of spectrin subunits to give heterodimes and tetamers. %%% Underneath the human red blood cell membranes is a skeletal network of proteins. This skeletal network provides many of the membrane properties, including mechanical and morphological properties, of the red cells. Abnormal skeletal networks often lead to abnormal red blood cells causing different types of anemia in patients. The major proteins in this network is called spectrin. Abnormal spectrin may lead to abnormal skeletal networks. Thus it is important to understand the structure of the spectrin molecule. However, spectrin is a very large protein molecule for detailed structural studies. In our project, we will synthesize a subfragment of the spectrin molecule, using recombinant DNA methods and bacterial cells, and use reporter group techniques to monitor the structural features of the molecule. In particular, we will synthesize the region of the molecule that will bind its partner subunit to provide interwoven molecules of two subunits (a dimer) that will then bind to another spectrin dimer to form the skeletal network underlying red blood cell membrane surfaces. With the spectrin fragment, we will attach a label which exhibits a signal in a electron paramagnetic resonance instrument. By following the signal changes under different experimental conditions, we will be able to extract molecular information on the spectrin molecule. With the understanding of the spectrin molecule, we will gain insights into the abnormalities of some o the red blood cells from anemia patients. ***

9407779我们建议了解人类红细胞光谱蛋白的结构 - 在与 - subunit形成正面关联时，以提供异二聚体和四聚体。 我们将使用谷胱甘肽-S-转移酶基因融合系统在大肠杆菌中使用cDNA和PCR方法来表达大肠杆菌中的特定subunit段。 为这项研究选择的频谱段将从亚基的N末端开始，并以序列的不同位置结束，从而给出各种长度的肽。 将对具有分离的 - 亚基的这些纯化的 - 重组肽进行结合研究，以选择具有功能特性的肽。 从功能性肽中，将使用傅立叶变换红外技术来选择具有类似于完整谱蛋白的二级结构的肽。 这些肽的最短肽将受到定向诱变的位置，以提供一组肽，每种肽都有一个半胱氨酸，但在序列中的不同位置处于不同位置，以通过Spin Label Electron parmagnetic Resonance方法获得拓扑信息。 在该项目结束时，我们预计将获得有关 - 贝米特的N末端区域前60个氨基酸的结构信息，这些氨基酸是头对头关联所需的。 我们认为，对 - 和 - 育种的头对头关联的理解是进一步理解谱子亚基的组装以给出异质和卫生机的基础。 人红细胞膜下方的下方是蛋白质的骨骼网络。 该骨骼网络提供了许多红细胞的膜特性，包括机械和形态学特性。异常的骨骼网络通常会导致红细胞异常，导致患者不同类型的贫血。 该网络中的主要蛋白质称为Spectrin。 异常光谱可能导致异常的骨骼网络。 因此，了解光谱分子的结构很重要。 但是，光谱是一种非常大的蛋白质分子，用于详细的结构研究。 在我们的项目中，我们将使用重组DNA方法和细菌细胞合成光谱分子的亚碎片，并使用报告基组技术来监测分子的结构特征。 特别是，我们将合成将结合其伴侣亚基的分子区域，以提供两个亚基（一个二聚体）的交织分子，然后将其结合到另一个光谱二聚体，形成红细胞膜表面的骨骼网络。 使用Spectrin片段，我们将附加一个标签，该标签在电子顺磁共振仪器中显示信号。 通过遵循不同实验条件下的信号变化，我们将能够在光谱分子上提取分子信息。 在了解谱分子的情况下，我们将洞悉贫血患者红细胞的某些异常。 ***