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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.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.

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.


Structure of a LCP. (A) LCP consists of water channels (shown as colored cross-sections) and a continuous 3D lipid bilayer that allows free diffusion of the reconstituted membrane protein molecules. (B) A detailed view of GPCR molecules in a LCP bilayer.
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Figure 4: Structure of a LCP. (A) LCP consists of water channels (shown as colored cross-sections) and a continuous 3D lipid bilayer that allows free diffusion of the reconstituted membrane protein molecules. (B) A detailed view of GPCR molecules in a LCP bilayer.

Mentions: The most used systems for crystallization of GPCRs are lipidic mesophases (reviewed in Yin et al., 2014; Caffrey, 2015). LCPs are bicontinuous liquid crystals composed of a single, curved lipidic bilayer that separates two continuous, non-contacting channels filled with water medium (Figure 4). As a lipidic component different monoacylglycerols are used and among them monoolein (MAG9.9) is the most common one (Caffrey, 2015). LCP forms by mixing e.g., monoolein with water solution containing a solubilized membrane protein in a 3:2 weight ratio at 20°C. In this process, a membrane protein gets incorporated into the stabilizing and native-like environment of a lipidic bilayer. In certain conditions LCP transforms into lipidic sponge phase, which essentially preserves its bicontinuous structure, but it is a true liquid and lacks the liquid-crystal properties. If the right crystallization conditions are encountered, both LCP and lipidic sponge phase support crystal nucleation and growth. The most GPCR crystal structures were obtained from crystallization in a lipidic mesophase. In almost all cases, a mixture of monoolein and cholesterol in 9:1 weight ratio was used, because – as already discussed – cholesterol makes GPCRs more stable, less conformationally flexible and thus increases probability of their crystallization. A notable exception is the β2-adrenoceptor–Gs protein complex (Rasmussen et al., 2011b), for which a mixture of MAG7.7 and cholesterol in a weight ratio of 9:1 was used instead of a monoolein–cholesterol mixture. Rationale for this is the fact that MAG7.7 forms LCP with water channels that are large enough to accommodate a relatively large heterotrimeric G protein in the macromolecular complex (Figure 1).


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

Milić D, Veprintsev DB - Front Pharmacol (2015)

Structure of a LCP. (A) LCP consists of water channels (shown as colored cross-sections) and a continuous 3D lipid bilayer that allows free diffusion of the reconstituted membrane protein molecules. (B) A detailed view of GPCR molecules in a LCP bilayer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Structure of a LCP. (A) LCP consists of water channels (shown as colored cross-sections) and a continuous 3D lipid bilayer that allows free diffusion of the reconstituted membrane protein molecules. (B) A detailed view of GPCR molecules in a LCP bilayer.
Mentions: The most used systems for crystallization of GPCRs are lipidic mesophases (reviewed in Yin et al., 2014; Caffrey, 2015). LCPs are bicontinuous liquid crystals composed of a single, curved lipidic bilayer that separates two continuous, non-contacting channels filled with water medium (Figure 4). As a lipidic component different monoacylglycerols are used and among them monoolein (MAG9.9) is the most common one (Caffrey, 2015). LCP forms by mixing e.g., monoolein with water solution containing a solubilized membrane protein in a 3:2 weight ratio at 20°C. In this process, a membrane protein gets incorporated into the stabilizing and native-like environment of a lipidic bilayer. In certain conditions LCP transforms into lipidic sponge phase, which essentially preserves its bicontinuous structure, but it is a true liquid and lacks the liquid-crystal properties. If the right crystallization conditions are encountered, both LCP and lipidic sponge phase support crystal nucleation and growth. The most GPCR crystal structures were obtained from crystallization in a lipidic mesophase. In almost all cases, a mixture of monoolein and cholesterol in 9:1 weight ratio was used, because – as already discussed – cholesterol makes GPCRs more stable, less conformationally flexible and thus increases probability of their crystallization. A notable exception is the β2-adrenoceptor–Gs protein complex (Rasmussen et al., 2011b), for which a mixture of MAG7.7 and cholesterol in a weight ratio of 9:1 was used instead of a monoolein–cholesterol mixture. Rationale for this is the fact that MAG7.7 forms LCP with water channels that are large enough to accommodate a relatively large heterotrimeric G protein in the macromolecular complex (Figure 1).

Bottom Line: 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.

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.