title: Successful development and use of a thermodynamic stability screen for optimizing the yield of nucleotide binding domains.
abstract: ATP sensitive potassium (KATP) channels consist of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). SUR proteins are members of the ATP-binding cassette superfamily of proteins. Binding of ATP to the Kir6.x subunit mediates channel inhibition, whereas MgATP binding and hydrolysis at the SUR NBDs results in channel opening. Mutations in SUR1 and SUR2A NBDs cause diseases of insulin secretion and cardiac disorders, respectively, underlying the importance of studying the NBDs. Although purification of SUR2A NBD1 in a soluble form is possible, the lack of long-term sample stability of the protein in a concentrated form has precluded detailed studies of the protein aimed at gaining a molecular-level understanding of how SUR mutations cause disease. Here we use a convenient and cost-effective thermodynamic screening method to probe stabilizing conditions for SUR2A NBD1. Results from the screen are used to alter the purification protocol to allow for significantly increased yields of the purified protein. In addition, the screen provides strategies for long-term storage of NBD1 and generating NBD1 samples at high concentrations suitable for NMR studies. NMR spectra of NBD1 with MgAMP-PNP are of higher quality compared to using MgATP, indicating that MgAMP-PNP be used as the ligand in future NMR studies. The screen presented here can be expanded to using different additives and can be employed to enhance purification yields, sample life times, and storage of other low stability nucleotide binding domains, such as GTPases.
authors.affiliation: Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada; Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd., Mississauga, Ontario L5L 1C6, Canada.
authors.forename: Elvin D
authors.initials: ED
authors.lastname: de Araujo
authors.affiliation: Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada; Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd., Mississauga, Ontario L5L 1C6, Canada; Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario M5S 3G5, Canada. Electronic address: voula.kanelis@utoronto.ca.
authors.forename: Voula
authors.initials: V
authors.lastname: Kanelis
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D054086:KATP Channels / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D064233:Sulfonylurea Receptors / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: C577510:ABCC9 protein, human; D004268:DNA-Binding Proteins; D054086:KATP Channels; D064233:Sulfonylurea Receptors; C092520:uK-ATP-1 potassium channel; D000255:Adenosine Triphosphate
keywords: ATP-sensitive (K(ATP)) potassium channel; Nuclear magnetic resonance (NMR) spectroscopy; Nucleotide binding domain (NBD); Osmolyte; Sulfonylurea receptor (SUR); Thermodynamic buffer screen