Expression and characterization of soluble amino-terminal domain of NR2B subunit of N-methyl-d-aspartate receptor
| Autor: | Pei Qi Lim, Fui-Mee Ng, Chye Yun Yu, Leng-Hiong Lim, Esther Wong, Chian-Ming Low, Stephen F. Traynelis |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
Protein Denaturation
Protein Folding Circular dichroism Protein subunit Molecular Sequence Data Protein Engineering Receptors N-Methyl-D-Aspartate Biochemistry Article Inclusion bodies Inhibitory Concentration 50 chemistry.chemical_compound Piperidines Ifenprodil Humans Histidine Amino Acid Sequence Binding site Receptor Molecular Biology Inclusion Bodies Chemistry Circular Dichroism Recombinant Proteins Protein Structure Tertiary Protein Subunits Solubility NMDA receptor Protein folding |
| Zdroj: | Protein Science. 14:2275-2283 |
| ISSN: | 1469-896X 0961-8368 |
| DOI: | 10.1110/ps.051509905 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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