Solution NMR spectroscopy of [α-
            <sup>15</sup>
            N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence

Klein‐Seetharaman, Judith; Reeves, Philip J.; Loewen, Michèle C.; Getmanova, E. V.; Chung, John Y. L.; Schwalbe, Harald; Wright, Peter E.; Khorana, H G

doi:10.1073/pnas.052713999

Cited by 62 publications

(57 citation statements)

References 27 publications

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“…Further, nuclear Overhauser effects were observed between certain pairs of 19 F labels (9). More recently, study of ␣-15 N-lysine-labeled rhodopsin by high-resolution heteronuclear NMR spectroscopy (10) showed that whereas motions were detected on the nanosecond time scale in the single lysine located in the C terminus ( Fig. 1 A), lysines present elsewhere in rhodopsin showed motions in micro-to millisecond time scales.…”

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confidence: 93%

Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR

Klein‐Seetharaman

Yanamala

Javeed

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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G protein-coupled receptors are cell-surface seven-helical membrane proteins that undergo conformational changes on activation. The mammalian photoreceptor, rhodopsin, is the beststudied member of this superfamily. Here, we provide the first evidence that activation in rhodopsin may involve differential dynamic properties of side-chain versus backbone atoms. Highresolution NMR studies of ␣-15 N-labeled receptor revealed large backbone motions in the inactive dark state. In contrast, indole side-chain 15 N groups of tryptophans showed well resolved, equally intense NMR signals, suggesting restriction to a single specific conformation.

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confidence: 93%

Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR

Klein‐Seetharaman

Yanamala

Javeed

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…16 However, in the GABA A R, much lower specific activities (1-6 pmol/ mg) have been reported. [17][18][19] In contrast, in the single subunit G protein-coupled receptor (GPCR) superfamily sufficient quantities of protein have been expressed in mammalian cells to allow structural information to be obtained in solution by electron paramagnetic resonance (EPR) 20 and nuclear magnetic resonance (NMR) spectroscopy 21 and recently by crystallography. 22 A promising approach has been the employment of an induction strategy analogous to that used in bacterial expression systems that allows the cells to grow normally to high density before the demand of high-level protein expression is imposed on them.…”

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confidence: 99%

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors

et al. 2010

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The human neuronal Cys-loop ligand-gated ion channel superfamily of ion channels are important determinants of human behavior and the target of many drugs. It is essential for their structural characterization to achieve high-level expression in a functional state. The aim of this work was to establish stable mammalian cell lines that enable high-level heterologous production of pure receptors in a state that supports agonist-induced allosteric conformational changes. In a tetracycline-inducible stable human embryonic kidney cells (HEK293S) cell line, GABA A receptors containing a1 and b3 subunits could be expressed with specific activities of 29-34 pmol/mg corresponding to 140-170 pmol/plate, the highest expression level reported so far. Comparable figures for serotonin (5-HT 3A ) receptors were 49-63 pmol/mg and 245-315 pmol/plate. The expression of 10 nmol of either receptor in suspension in a bioreactor required 0.3-3.0 L. Both receptor constructs had a FLAG epitope inserted at the N-terminus and could be purified in one Abbreviations: 5-HT 3 AR, 5-hydroxytryptamine-3A receptor; CMC, critical micelle concentration; DDM, n-dodecyl-b-D-maltopyranoside; EPR, electron paramagnetic resonance; GABA A a1b3R, gamma-aminobutyric acid type A receptor with a1 and b3 subunits; GPCR, G protein-coupled receptor; GlyR, glycine receptor; HEK293, human embryonic kidney cells; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; NMR, nuclear magnetic resonance; PEG, poly(ethylene glycol).Additional Supporting Information may be found in the online version of this article. Published by Wiley-Blackwell. V C 2010 The Protein Society step after solubilization using ANTI-FLAG affinity chromatography with yields of 30-40%. Purified receptors were functional. Binding of the agonist [ 3 H]muscimol to the purified GABA A R was enhanced allosterically by the general anesthetic etomidate, and purified 5-hydroxytryptamine-3A receptor supported serotonin-stimulated cation flux when reconstituted into lipid vesicles.

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“…To meet these needs, stable mammalian cell lines for high-level expression of the opsin gene and its mutants were developed (1,2). The application of NMR spectroscopy to structure-function studies of rhodopsin thus became possible (3)(4)(5)(6). Recently, evidence has been accumulating that certain gene products either fail to be expressed in adequate amounts in the constitutive expression systems above or that viable stable cell lines cannot be generated for certain mutant opsin genes.…”

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confidence: 99%

Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants

Reeves

Kim

Khorana

2002

Proc. Natl. Acad. Sci. U.S.A.

192

196

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Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg͞liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate. The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing tetO sequences as well as the neomycin resistance gene under control of the weak H 2L d promoter. The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products. By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q͞E134Q͞M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium.G protein-coupled receptors ͉ HEK293S cells ͉ cytomegalovirus promoter ͉ glycosylation ͉ sodium butyrate C ertain studies on rhodopsin and its mutants require relatively large amounts of purified proteins. To meet these needs, stable mammalian cell lines for high-level expression of the opsin gene and its mutants were developed (1, 2). The application of NMR spectroscopy to structure-function studies of rhodopsin thus became possible (3-6). Recently, evidence has been accumulating that certain gene products either fail to be expressed in adequate amounts in the constitutive expression systems above or that viable stable cell lines cannot be generated for certain mutant opsin genes. For example, stable cell lines carrying the rhodopsin kinase gene expressed the kinase at levels much lower than those predicted from the transient transfection experiments (7). Further, we failed to isolate a viable stable cell line for expression of the strongly constitutively active opsin mutant, E113Q͞E134Q͞M257Y. It seems likely that the proteins produced by unregulated expression of the genes above are cytotoxic and, indeed, in certain cases may be lethal. To overcome these difficulties, we wanted to develop a strategy frequently used in bacterial expression systems (8, 9) in which membrane proteins toxic to the bacterial cells are expressed by induction of the desired gene only after achieving growth of cells to nearmaximum density. Previously, this strategy enabled large-scale production in Escherichia coli of numerous toxic bacterio-opsin mutants (10). Therefore, our present aim was to develop an inducible expression system in stable mammalian cell lines.A tetracycline (tet)-regulated gene expression system in mammalian cells was first described by Gossen and Bujard (11). By fusing the bacterial tet repressor (TetR) with the activating domain of the viral protein 16 (VP-16), a tetracycline-controlled transactivator was generated that stimulated transcription of a desired gene from a minimal cytomegalovirus (CMV) promo...

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Solution NMR spectroscopy of [α- ¹⁵ N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence

Cited by 62 publications

References 27 publications

Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR

Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors

Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants

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Solution NMR spectroscopy of [α- 15 N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence

Cited by 62 publications

References 27 publications

Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR

Differential dynamics in the G protein-coupled receptor rhodopsin revealed by solution NMR

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABAA and serotonin receptors

Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants

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Solution NMR spectroscopy of [α- ¹⁵ N]lysine-labeled rhodopsin: The single peak observed in both conventional and TROSY-type HSQC spectra is ascribed to Lys-339 in the carboxyl-terminal peptide sequence

High‐level expression and purification of Cys‐loop ligand‐gated ion channels in a tetracycline‐inducible stable mammalian cell line: GABA_A and serotonin receptors