Biochemical and Biophysical Studies of Human SUR1 NBD1, Rat SUR2A NBD2 and the Role of the C-terminal Extension in Rat SUR2A NBD1

• Main Author: Alvarez, Claudia Paola
• Other Authors: Voula, Kanelis
• Language: en_ca
• Published: 2013
• Subjects: rSUR2A NBD1; hSUR1 NBD1; rSUR2A NBD2; NBDs; potassium channel; KATP channel; NBD1 phosphorylation; C-terminal extension; Tryptophan quenching; Thermal denaturation; NMR; Regulation of NBD1; Acrylamide quenching; Iodide quenching; Fluorescence; Circular dichroism; NBD1 C-terminal tail; MgATP binding; 0487
• Online Access: http://hdl.handle.net/1807/35107

SUR2A-mediated regulation of KATP channels is affected by residues belonging to the C terminus of the first nucleotide binding domain (NBD1). We studied the C-terminal region of NBD1 by comparing experiments using NBD1 S615-D914 and NBD1 S615-K972 constructs to studies of NBD1 S615-L933 also performed in our laboratory. Our NMR data suggests that the C-terminal region of NBD1 from residues Q915 to L933 is disordered and transiently contacts the NBD1 core, which may affect NBD1 phosphorylation. Tryptophan quenching fluorescence experiments corroborate that the Q915-L933 C-terminal tail contacts the NBD1 core. Fluorescence thermal denaturation experiments suggest that NBD1 S615-D914 has a higher affinity for MgATP compared with NBD1 S615-L933, implying that the C-terminal tail varies MgATP binding. Additional experiments were performed to identify soluble constructs of hSUR1 NBD1 and rSUR2A NBD2 that would allow detailed biophysical studies of these domains. Some of the constructs studied showed improved solubility and stability.