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Large-scale production and protein engineering of G protein-coupled receptors for structural studies.

Milić D, Veprintsev DB - Front Pharmacol (2015)

Bottom Line: Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology.On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities.In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen Switzerland.

ABSTRACT
Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures.

No MeSH data available.


Detergents used in solubilization and purification of rhodopsin and other GPCRs for crystallographic studies.
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Figure 3: Detergents used in solubilization and purification of rhodopsin and other GPCRs for crystallographic studies.

Mentions: A rather robust GPCR, rhodopsin, could be solubilized from bovine ROS membranes without losing its structural integrity in a variety of detergents (Figure 3): a mixture of HPTO and NG (Okada et al., 1998, 2000; Salom et al., 2006a), OG (Park et al., 2008, 2013; Scheerer et al., 2008; Choe et al., 2011), short-chain HTG (Okada et al., 2004), zwitter-ionic LDAO (Edwards et al., 2004) and a mixture of OG and mild DDM (Choe et al., 2011). Bovine rhodopsin mutants produced in mammalian cells were solubilized in DM (Standfuss et al., 2007) or DDM (Standfuss et al., 2011; Deupi et al., 2012; Singhal et al., 2013).


Large-scale production and protein engineering of G protein-coupled receptors for structural studies.

Milić D, Veprintsev DB - Front Pharmacol (2015)

Detergents used in solubilization and purification of rhodopsin and other GPCRs for crystallographic studies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379943&req=5

Figure 3: Detergents used in solubilization and purification of rhodopsin and other GPCRs for crystallographic studies.
Mentions: A rather robust GPCR, rhodopsin, could be solubilized from bovine ROS membranes without losing its structural integrity in a variety of detergents (Figure 3): a mixture of HPTO and NG (Okada et al., 1998, 2000; Salom et al., 2006a), OG (Park et al., 2008, 2013; Scheerer et al., 2008; Choe et al., 2011), short-chain HTG (Okada et al., 2004), zwitter-ionic LDAO (Edwards et al., 2004) and a mixture of OG and mild DDM (Choe et al., 2011). Bovine rhodopsin mutants produced in mammalian cells were solubilized in DM (Standfuss et al., 2007) or DDM (Standfuss et al., 2011; Deupi et al., 2012; Singhal et al., 2013).

Bottom Line: Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology.On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities.In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biomolecular Research, Paul Scherrer Institut, Villigen Switzerland.

ABSTRACT
Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled receptors and their low long-term stabilities. In this review, we discuss methods used for expression and purification of GPCRs for crystallographic and NMR studies. We also summarize protein engineering methods that played a crucial role in obtaining GPCR crystal structures.

No MeSH data available.