NMR paper Structural characterization of triple transmembrane domain containing fragments of a yeast G protein-coupled receptor in an organic : aqueous environment by solution-state NMR spectroscopy.

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[NMR paper] Structural characterization of triple transmembrane domain containing fragments of a yeast G protein-coupled receptor in an organic : aqueous environment by solution-state NMR spectroscopy.

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NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

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TALOS

Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

PECAN

Flexibility from chemical shifts:

RCI

Interactions from chemical shifts:

HADDOCK

Chemical shifts re-referencing:

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NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

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MestReS

V-NMR

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Backbone S2

Methyl S2

B-factor

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Shiftx2

Sparta+

Camshift

CH3shift- Methyl

ArShift- Aromatic

ShiftS

Proshift

PPM

CheShift-2- Cα

From sequence:

Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

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Protein disorder:

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Protein solubility:

Isotope labeling:

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02-04-2015, 09:57 PM

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Structural characterization of triple transmembrane domain containing fragments of a yeast G protein-coupled receptor in an organic : aqueous environment by solution-state NMR spectroscopy.

Structural characterization of triple transmembrane domain containing fragments of a yeast G protein-coupled receptor in an organic : aqueous environment by solution-state NMR spectroscopy.

Related Articles Structural characterization of triple transmembrane domain containing fragments of a yeast G protein-coupled receptor in an organic : aqueous environment by solution-state NMR spectroscopy.

J Pept Sci. 2015 Feb 2;

Authors: Fracchiolla KE, Cohen LS, Arshava B, Poms M, Zerbe O, Becker JM, Naider F

Abstract
This report summarizes recent biophysical and protein expression experiments on polypeptides containing the N-terminus, the first, second, and third transmembrane (TM) domains and the contiguous loops of the ?-factor receptor Ste2p, a G protein-coupled receptor. The 131-residue polypeptide Ste2p(G31-R161), TM1-TM3, was investigated by solution NMR in trifluoroethanol/water. TM1-TM3 contains helical TM domains at the predicted locations, supported by continuous sets of medium-range NOEs. In addition, a short helix N-terminal to TM1 was detected, as well as a short helical stretch in the first extracellular loop. Two 161-residue polypeptides, [Ste2p(M1-R161), NT-TM1-TM3], that contain the entire N-terminal sequence, one with a single mutation, were directly expressed and isolated from Escherichia coli in yields as high as 30 mg/L. Based on its increased stability, the L11P mutant will be used in future experiments to determine long-range interactions. The study demonstrated that 3-TM domains of a yeast G protein-coupled receptor can be produced in isotopically labeled form suitable for solution NMR studies. The quality of spectra is superior to data recorded in micelles and allows more rapid data analysis. No tertiary contacts have been determined, and if present, they are likely transient. This observation supports earlier studies by us that secondary structure was retained in smaller fragments, both in organic solvents and in detergent micelles, but that stable tertiary contacts may only be present when the protein is imbedded in lipids. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

PMID: 25645975 [PubMed - as supplied by publisher]

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