The objective of this project is to establish that residues 696-894 of the hamster sulfonylurea receptor (SUR1) encode a nucleotide binding domain (NBF1). Furthermore, we hypothesize binding and hydrolysis of Mg- ATP by NBF1 has structural consequences which will help understand the mechanism of SUR1 triggered insulin release. Lastly, we propose NBF1 may also contain sulfonylurea binding sites which affect its nucleotide binding and hydrolysis ability.
Aim I is to design, express and purify to homogeneity residues 696-894 of SUR1.
Aim II is to characterize the nucleotide and sulfonylurea binding ability of-NBF1 using intrinsic tryptophan fluorescence, isothermal titration calorimetry and site specific NMR.
Aim III will further-characterize NBF1 by establishing its ATPase activity. Finally, Aim IV is to define the structural and dynamic properties of the nucleotide binding reaction using solution-state NMR. This study will provide potential mechanisms for sulfonylurea action in SUR1 as well as structurally explain genetic variations in SUR1 in relation to diabetes.
