| The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor of the immunoglobulin superfamily. RAGE is composed of three extracellular domains followed by a single-transmembrane and a short cytoplasmic domain (ctRAGE). RAGE is linked to many chronic inflammatory diseases including diabetes, Alzheimer's disease, tumors, and atherosclerosis. Upon binding to one of its various ligands including AGEs, amyloid-beta, HMGB1, and S100/calgranulins, the cytoplasmic domain of RAGE binds to the formin DIAPH1 activating intracellular signaling. This intracellular domain would therefore be a likely target for drug development.;Mutations were made to ctRAGE in order to determine the ctRAGE-DIAPH1 binding region. The amino acids W2, Q3, Q6, E11, and R12 were mutated to alanine residues for a total of five different mutants. The proteins were amplified and purified before combining with a series of small molecules in order to determine binding affinity. No binding to the small molecules would indicate that the mutations made to the cytoplasmic domain were located within the binding region.;Fluorescence spectroscopy using tryptophan residues was utilized to measure the dissociation constants (Kd) after combining the mutants with small molecules. Large Kd values indicate no affinity for the small molecule and therefore no binding. NMR characterization of the mutants was conducted by collecting 1H{15N}-HSQC NMR spectra of each mutant as well as the mutant combined with a small molecule. Peak shift mapping was completed to determine if binding occurred. A peak shift greater than 0.05 ppm would indicate binding between protein and small molecule. The five mutations made to ctRAGE were found to have destroyed the binding site of the receptor. |
