title: Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain.
abstract: The ionotropic glutamate receptors (GluR) are the primary mediators of excitatory synaptic transmission in the brain. GluR agonist binding has been localized to an extracellular domain whose core is homologous to the bacterial periplasmic binding proteins (PBP). We have established routine, baculovirus-mediated expression of a complete ligand-binding domain construct at the 10-L scale, yielding 10-40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connect the domain core to the transmembrane domains of the channel and to the N-terminal 'X'-domain. These linker peptides have been implicated in modulating channel physiology. Such extended constructs have proven difficult to express in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the domain involves a substantial heat capacity change and that at physiological temperatures, the reaction is both entropically and enthalpically favorable.
authors.affiliation: Ion Channel Structure Research Group, Max Planck Institute for Medical Research, Heidelberg, Germany. madden@mpimf-heidelberg.mpg.de
authors.forename: D R
authors.initials: DR
authors.lastname: Madden
authors.forename: R
authors.initials: R
authors.lastname: Abele
authors.forename: A
authors.initials: A
authors.lastname: Andersson
authors.forename: K
authors.initials: K
authors.lastname: Keinänen
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002478:Cells, Cultured; D002850:Chromatography, Gel; D018698:Glutamic Acid / Q000378:metabolism*; D008970:Molecular Weight; D017470:Receptors, Glutamate / Q000096:biosynthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D018411:Spodoptera; D013816:Thermodynamics
chemical_list: D017470:Receptors, Glutamate; D011994:Recombinant Proteins; D018698:Glutamic Acid