Crystal Structure of a Thermally Stable Rhodopsin Mutant
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- 作者：Jö ; rg St ; fuss ; Guifu Xie ; Patricia C. Edwards ; Manfred Burghammer ; Daniel D. Oprian ; Gebhard F.X. Schertler
- 关键词：G protein-coupled receptors ; signal transduction ; visual system ; rhodopsin ; retinitis pigmentosa
- 刊名：Journal of Molecular Biology
- 出版年：2007
- 出版时间：5 October 2007
- 年：2007
- 卷：372
- 期：5
- 页码：1179-1188
- 全文大小：1127 K
文摘
We determined the structure of the rhodopsin mutant N2C/D282C expressed in mammalian cells; the first structure of a recombinantly produced G protein-coupled receptor (GPCR). The mutant was designed to form a disulfide bond between the N terminus and loop E3, which allows handling of opsin in detergent solution and increases thermal stability of rhodopsin by 10 deg.C. It allowed us to crystallize a fully deglycosylated rhodopsin (N2C/N15D/D282C). N15 mutations are normally misfolding and cause retinitis pigmentosa in humans. Microcrystallographic techniques and a 5 μm X-ray beam were used to collect data along a single needle measuring 5 μm × 5 μm × 90 μm. The disulfide introduces only minor changes but fixes the N-terminal cap over the β-sheet lid covering the ligand-binding site, a likely explanation for the increased stability. This work allows structural investigation of rhodopsin mutants and shows the problems encountered during structure determination of GPCRs and other mammalian membrane proteins.