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Structure. 2014 Nov 4;22(11):1657-64. doi: 10.1016/j.str.2014.08.022. Epub 2014 Oct 23.

Modified T4 Lysozyme Fusion Proteins Facilitate G Protein-Coupled Receptor Crystallogenesis.

Author information

1
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305, USA.
2
Department of Chemical and Systems Biology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305, USA.
3
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305, USA; Departments of Structural Biology and Molecular and Cellular Physiology, Stanford University School of Medicine, 299 Campus Drive, Stanford, CA 94305, USA.
4
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305, USA. Electronic address: kobilka@stanford.edu.

Abstract

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters. Most GPCR crystal structures have been obtained using a fusion protein strategy where the flexible third intracellular loop is replaced by T4 lysozyme (T4L). However, wild-type T4L may not be ideally suited for all GPCRs because of its size and the inherent flexibility between the N- and C-terminal subdomains. Here we report two modified T4L variants, designed to address flexibility and size, that can be used to optimize crystal quality or promote alternative packing interactions. These variants were tested on the M3 muscarinic receptor (M3). The original M3-T4L fusion protein produced twinned crystals that yielded a 3.4 Å structure from a 70 crystal data set. We replaced T4L with the modified T4L variants. Both T4L variants yielded M3 muscarinic receptor crystals with alternate lattices that were not twinned, including one that was solved at 2.8 Å resolution.

PMID:
25450769
PMCID:
PMC4408211
DOI:
10.1016/j.str.2014.08.022
[Indexed for MEDLINE]
Free PMC Article

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