Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation
| Autor: | Christopher G. Tate, Byron Carpenter |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Models Molecular GTPase-activating protein Receptor Adenosine A2A G protein Bioengineering GTPase Biology Protein Engineering Biochemistry RS Gs 03 medical and health sciences GPCR Protein Domains GTP-Binding Proteins Heterotrimeric G protein G protein-coupled receptor Molecular Biology mini-Gs Binding Sites Protein Stability Rational design Temperature QP Peptide Fragments mini G protein G beta-gamma complex 030104 developmental biology Mutagenesis Biophysics Original Article Signal transduction Crystallization complex Biotechnology |
| Zdroj: | Protein Engineering, Design and Selection |
| ISSN: | 1741-0126 |
| Popis: | G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs. Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation. |
| Databáze: | OpenAIRE |
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