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Opella, Stanley J.; Marassi, Francesca M.; Gesell, Jennifer J.; Valente, Ana Paula; Kim, Y.; Oblatt-Montal, M.; Montal, Mauricio

doi:10.1038/7610

Cited by 373 publications

(372 citation statements)

References 39 publications

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“…50% of the total protein, the ␣-helical content of about 25% derived from our CD and FTIR-data would correspond to a portion of about 12% of the full-length ␣1 subunit. Given that the helical structure of TM2 (8,9,15,40) contributes a further 5%, a total of at least 17% ␣-helical structure would be expected for the entire GlyR ␣1 subunit. This is in qualitative agreement with the prediction of about 15% ␣-helical content for the reconstituted GlyR ␣1-subunit (41) or its fragment ␣1-(165-291) (42).…”

Section: Functional Refolding Of Isolated Glyr Domainsmentioning

confidence: 99%

“…A large cytosolic loop, flanked by TM3 and TM4, is thought to mediate intracellular interactions. Based on NMR studies of corresponding synthetic peptides, TM2 of the nAChR and GlyR has been demonstrated to be ␣-helical (8,9). Several attempts have been made to elucidate the structure of the nAChR extracellular domain, which is widely seen as a prototype of the structural homologous GlyR.…”

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confidence: 99%

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Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

Breitinger

Bauer

Fahmy

et al. 2004

Journal of Biological Chemistry

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The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding ␣-and gephyrin anchoring ␤-subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (␣1-(1-219)) and the large intracellular TM3-4 loop (␣1-(309 -392)) of the human GlyR ␣1-subunit were individually expressed in HEK293 cells and in Escherichia coli. The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested ϳ25% ␣-helix and ϳ48% ␤-sheet for the extracellular domain, while no ␣-helices were detectable for the TM3-4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli, we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of ϳ5 and 15 subunits. Likewise, for the intracellular domain from E. coli, a single molecular mass peak of ϳ49 kDa indicated oligomerization in a defined native structure. As shown by [ 3 H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.The inhibitory glycine receptor (GlyR) 1 is a neurotransmitter-gated ion channel, mediating rapid synaptic transmission in the central nervous system (1-3). The GlyR is a member of the ligand-gated ion channel superfamily, which also includes the nicotinic acetylcholine receptor (nAChR), serotonin 5-HT 3 receptor, as well as GABA A/C receptors. Members of this protein family share a quaternary structure of five subunits surrounding a central ion-conducting pore (4 -6). The GlyR isoform prevalent in adult mammalian central nervous system is thought to comprise three ␣1-subunits and two structural ␤-subunits, which are thought to contribute to synaptic anchoring of receptor complexes. Generally, GlyR ␣-subunits possess the ability to form functional homomeric receptor channels. The subunit topology of the nAChR superfamily, as deduced from hydropathy analysis, consists of a large N-terminal extracellular domain, followed by four hydrophobic stretches of sufficient length to span the plasma membrane, and the extracellular C terminus (7). Of all transmembrane regions, TM2 forms the inner lining of the central ion channel. A large cytosolic loop, flanked by TM3 and TM4, is thought to mediate intracellular interactions.

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Section: Functional Refolding Of Isolated Glyr Domainsmentioning

confidence: 99%

mentioning

confidence: 99%

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

Breitinger

Bauer

Fahmy

et al. 2004

Journal of Biological Chemistry

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“…Recent developments in bacterial expression systems for the preparation of recombinant isotopically labeled membrane proteins, methods for sample preparation, pulse sequences for high-resolution spectroscopy, and structural indices that guide the structure assembly process, have greatly extended the capabilities of these techniques, and the structures of a variety of helical membrane proteins have been determined by NMR in micelles and in bilayers [3][4][5][6][7][8][9][10][11].…”

Section: Introductionmentioning

confidence: 99%

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

Franzin

Gong

Thai

et al. 2007

Methods

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Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na, KATPase.

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“…The effect of binding to DPC micelles and lipid II on the mersacidin dynamics are characterized by means of 15 N relaxation together with gradient-edited diffusion experiments. Despite the large number of published solution NMR structural and relaxation dynamics studies of membrane proteins and peptides (12)(13)(14)(15)(16), only a few examples of high resolution NMR studies of protein/peptide-ligand interactions in the presence of membrane-like environments are available to date (17,18). We will show that the differences in sample environments result in substantial conformational changes that modulate the charge accessibility.…”

Section: In Dodecylphosphocholine (Dpc)mentioning

confidence: 99%