Hori,T., Nakamura,M., Yokomizo,T., Shimizu,T., and Miyano,M.
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In this study, we introduced structure-based rational mutations in the guinea pig leukotriene B4 receptor
(gpBLT1) in order to enhance the stabilization of the protein. Elements thought to be unfavorable for the
stability of gpBLT1 were extracted based on the stabilization elements established in soluble proteins,
determined crystal structures of G-protein-coupled receptors (GPCRs), and multiple sequence alignment. The two unfavorable residues His832.67 and Lys883.21, located at helix capping sites, were replaced with Gly (His83Gly2.67 and Lys88Gly3.21). The modi ed protein containing His83Gly2.67/Lys88Gly3.21 was highly expressed, solubilized, and puri ed and exhibited improved thermal stability by 4°C in comparison with that of the original gpBLT1 construct. Owing to the double mutation, the expression level increased by 6-fold (Bmax ¼311 pmol/mg) in the membrane fraction of Pichia pastoris. The ligand binding afnity was similar to that of the original gpBLT1 without the mutations. Similar unfavorable residues have been observed at helix capping sites in many other GPCRs; therefore, the replacement of such residues with more favorable residues will improve stabilization of the GPCR structure for the crystallization.
Research papers (academic journals)
