Abstract
Metabotropic glutamate receptors (mGluRs) are involved in the regulation of many physiological and pathological processes in the central nervous system. The extracellular domain (ECD) of mGluR subtype 3 (mGluR3) was produced using the baculovirus expression system and purified from the culture medium. However, the recombinant protein showed heterogeneity in molecular weight on SDS-PAGE analysis. It was found that the unglycosylation of Asn414 significantly reduced the heterogeneity. Consequently, three site-specifically unglycosylated mutant proteins of mGluR3 ECD, replacing Asn414 only or replacing Asn414 in combination with other glycosylation sites, were successfully crystallized in the presence of L-glutamate. Among them, crystals of the N414/439Q mutant diffracted X-rays to 2.35 A resolution using synchrotron radiation. The crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a = 84.0, b = 97.5, c = 108.1 A, beta = 93.0 degrees . Assuming the presence of two protomers per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 3.5 A(3) Da(-1) and the solvent content was 65%.