Development and crystallization of a minimal thermostabilised G protein-coupled receptor
Section snippets
Removal of flexible regions in β1AR
It is generally considered that flexible regions in a protein might prevent the growth of well-ordered crystals and that it might be beneficial to remove them, either by site-directed mutagenesis or by limited proteolysis of the purified protein. An alignment of the β-adrenergic receptors (not shown) clearly indicated three areas that were non-conserved and thus potentially represented regions that might be flexible and redundant for ligand binding, namely the N-terminus, the C-terminus and
Discussion
No three dimensional crystals of the full-length, non-stabilised β1AR6 construct were ever been obtained from sitting or hanging drop crystallization trials, despite the high expression level, the purity of the preparation, the steps taken to reduce heterogeneity due to post-translational modifications and the implementation of large-scale robotic crystallization screening. We ascribe this to three major problems, namely receptor flexibility, heterogeneity of signalling states and poor
Materials
Sf9 and Tni (High 5™) cells were obtained from Invitrogen, the baculovirus transfer vector pBacPAK8 was from BD Clontech, and linearized baculovirus DNA (Baculogold™) was from Pharmingen. All detergents were from Anatrace, with the exception of LDAO that was from Fluka and C8E4 that was from Bachem. Alprenolol sepharose CL-4B was synthesized as described previously [29]. All other chromatographic equipment, columns and materials were from GE Healthcare, PEGs were from Fluka, (−)-alprenolol and (
Acknowledgments
This work was supported by a joint grant from Pfizer Global Research and Development and from the MRCT Development Gap Fund, in addition to core funding from MRC. G.F.X.S. was financially supported by a Human Frontier Science Project (HFSP) programme grant (RG/0052), a European Commission FP6 specific targeted research project (LSH-2003-1.1.0-1) and an ESRF long-term proposal. We also thank C. Riekel and M. Burghammer at ID13, D. Flot and S. McSweeney at ID 23.2, which are both microfocus
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