Purified DAMGO-bound μOR was mixed with a 1.2 molar excess of
Gi heterotrimer. The coupling reaction was allowed to proceed at
24 °C for 1 hour and was followed by addition of apyrase to catalyze
hydrolysis of unbound GDP, which destabilizes the nucleotide-free
complex40 . After one
more hour at 25 °C, a 4-fold volume of 20 mM Hepes pH 7.5, 100 mM NaCl,
1% lauryl maltose neopentyl glycol (L-MNG), 0.1% CHS was added
to the complexing reaction to initiate detergent exchange. After one hour
incubation at 25 °C to allow micelle exchange, 1 mM MnCl2 and
lambda phosphatase (New England Biolabs) were added to dephosphorylate the
preparation. This reaction was further incubated at 4 °C for 2 hours. To
remove excess G protein and residual DDM, the complexing mixture was purified by
M1 anti-FLAG affinity chromatography. Bound complex was first washed in a buffer
containing 1% L-MNG, followed by washes in gradually decreasing L-MNG
concentrations. The complex was then eluted in 20mM Hepes pH 7.5, 100mM NaCl,
0.01% MNG/0.001% CHS, 300 nM DAMGO, 5 mM EDTA, and FLAG peptide.
The eluted complex was supplemented with 100 μM TCEP to provide a
reducing environment. The tobacco etch virus (TEV) protease and human rhinovirus
3C protease were added to cleave the flexible μOR amino- and carboxy-
termini. Finally, a 1.2 molar excess of scFv16 was added to the preparation.
Once cleavage of the termini was confirmed by SDS-PAGE, the
μOR-Gi-scFv16 complex was purified by size exclusion
chromatography on a Superdex 200 10/300 column in 20mM Hepes pH 7.5, 100mM NaCl,
300 nM DAMGO, 0.00075% MNG and 000025% GDN. Peak fractions were
concentrated to ~7 mg/mL for electron microscopy studies.
Gi heterotrimer. The coupling reaction was allowed to proceed at
24 °C for 1 hour and was followed by addition of apyrase to catalyze
hydrolysis of unbound GDP, which destabilizes the nucleotide-free
complex40 . After one
more hour at 25 °C, a 4-fold volume of 20 mM Hepes pH 7.5, 100 mM NaCl,
1% lauryl maltose neopentyl glycol (L-MNG), 0.1% CHS was added
to the complexing reaction to initiate detergent exchange. After one hour
incubation at 25 °C to allow micelle exchange, 1 mM MnCl2 and
lambda phosphatase (New England Biolabs) were added to dephosphorylate the
preparation. This reaction was further incubated at 4 °C for 2 hours. To
remove excess G protein and residual DDM, the complexing mixture was purified by
M1 anti-FLAG affinity chromatography. Bound complex was first washed in a buffer
containing 1% L-MNG, followed by washes in gradually decreasing L-MNG
concentrations. The complex was then eluted in 20mM Hepes pH 7.5, 100mM NaCl,
0.01% MNG/0.001% CHS, 300 nM DAMGO, 5 mM EDTA, and FLAG peptide.
The eluted complex was supplemented with 100 μM TCEP to provide a
reducing environment. The tobacco etch virus (TEV) protease and human rhinovirus
3C protease were added to cleave the flexible μOR amino- and carboxy-
termini. Finally, a 1.2 molar excess of scFv16 was added to the preparation.
Once cleavage of the termini was confirmed by SDS-PAGE, the
μOR-Gi-scFv16 complex was purified by size exclusion
chromatography on a Superdex 200 10/300 column in 20mM Hepes pH 7.5, 100mM NaCl,
300 nM DAMGO, 0.00075% MNG and 000025% GDN. Peak fractions were
concentrated to ~7 mg/mL for electron microscopy studies.
