Our broad, long-term objectives are to understand conformations of critical regions in various spectrin isoforms and to correlate specific structural features in these isoforms with functions unique to individual isoforms. We begin by studying the association affinities and local structures at the tetramerization site of two different spectrins, using recombinant peptide model systems. The four model peptides to be used are Spalpha-1-368 (erythrocyte), SpalphaII-1-359 (brain), SpbetaI-1689-2083 (erythrocyte) and SpbetaII-1697-2091 (brain). We have already determined the junction region conformation of SpalphaI-1-368. The junction region conformations of the other three peptides will be determined in this project by site-directed spin labeling EPR methods. Other biophysical methods will be used to ensure the integrity of the peptides. The association affinity will also be determined to correlate with the junction structural properties in these peptides. Alternate studies are proposed if the first part of this project shows that the junction regions play no role in regulating association affinities.
The specific aims are (1) To characterize the junction region conformation for Spalphall-1-359, (2) To determine association affinities of Spalpha-1-368 and SpalphaII-1-359 with SpbetaI-1689-2083 and with SpbetaII-1697-2091, (3) To characterize the junction region conformations in SpbetaI-1689-2083 and in SpbetaII-1697-2091, (4) To characterize the alpha- and beta-junction region conformations and interactions in the alphabeta complex for both alphaIbetaI and alphaIIbetaII systems, and (5) To identify key residues in SpalphaII-1-359 that affect its association affinity with SpbetaI-1689-2083 and with SpbetaII-1697-2091. Findings from these studies will provide insight toward developing molecular understanding of normal physiology involving spectrin molecules in erythrocytes, brain cells and other types of cells, and of diseases related to spectrin tetramers, such as hereditary hemolytic anemia, and neurological disorders. We believe that new disease markers and drug targets can be identified to help design products to prevent, diagnose and treat diseases through functional proteomic studies of spectrin isoforms and their protein-protein interactions. In addition to the spectrin systems, the information obtained from this study will also provide insight toward a better understanding of naturally occurring coiled-coil subunit-subunit associations.
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