title: Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium exchange to characterize allosteric regulation.
abstract: There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g., active and allosteric sites). Both nuclear magnetic resonance (NMR), by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H(2)O and D(2)O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D(2)O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D(2)O, but the effector continues to elicit an allosteric response.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, MS 3030, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
authors.forename: Charulata B
authors.initials: CB
authors.lastname: Prasannan
authors.forename: Antonio
authors.initials: A
authors.lastname: Artigues
authors.forename: Aron W
authors.initials: AW
authors.lastname: Fenton
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D003903:Deuterium / Q000032:analysis*; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D011817:Rabbits; D013816:Thermodynamics
chemical_list: D006859:Hydrogen; D003903:Deuterium