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. ***
