title: Expression and characterization of soluble amino-terminal domain of NR2B subunit of N-methyl-D-aspartate receptor.
abstract: N-methyl-D-aspartate (NMDA) receptors are involved in mediating excitatory synaptic transmissions in the brain and have been implicated in numerous neurologic disorders. The proximal amino-terminal domains (ATDs) of NMDA receptors constitute many modulatory binding sites that may serve as potential drug targets. There are few biochemical and structural data on the ATDs of NMDA receptors, as it is difficult to produce the functional proteins. Here an optimized method was established to reconstitute the insoluble recombinant ATD of NMDA receptor NR2B subunit (ATD2B) through productive refolding of 6xHis-ATD2B protein from inclusion bodies. Circular dichroism and dynamic light scattering characterizations revealed that the solubilized and refolded 6xHis-ATD2B adopted well-defined secondary structures and monodispersity. More significantly, the soluble 6xHis-ATD2B specifically bound ifenprodil to saturation. Ifenprodil bound to 6xHis-ATD2B with a dissociation constant (KD) of 127.5+/-45 nM, which was within the range of the IC50 determined electrophysiologically. This is the first report on a functional recombinant ATD2B with a characterized KD.
authors.affiliation: Glutamate Receptor Laboratory, National Neuroscience Institute, S308433, Singapore.
authors.forename: Esther
authors.initials: E
authors.lastname: Wong
authors.forename: Fui-Mee
authors.initials: FM
authors.lastname: Ng
authors.forename: Chye-Yun
authors.initials: CY
authors.lastname: Yu
authors.forename: Peiqi
authors.initials: P
authors.lastname: Lim
authors.forename: Leng-Hiong
authors.initials: LH
authors.lastname: Lim
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Traynelis
authors.forename: Chian-Ming
authors.initials: CM
authors.lastname: Low
mesh_terms: D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods; D002942:Circular Dichroism; D006639:Histidine / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry; D020128:Inhibitory Concentration 50; D008969:Molecular Sequence Data; D010880:Piperidines / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility
chemical_list: C121001:NR2B NMDA receptor; D010880:Piperidines; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins; D006639:Histidine; C010739:ifenprodil