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Global fold of human cannabinoid type 2 receptor probed by solid‐state 13 C‐, 15 N‐MAS NMR and molecular dynamics simulations
Author(s) -
Kimura Tomohiro,
Vukoti Krishna,
Lynch Diane L.,
Hurst Dow P.,
Grossfield Alan,
Pitman Michael C.,
Reggio Patricia H.,
Yeliseev Alexei A.,
Gawrisch Klaus
Publication year - 2014
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.24411
Subject(s) - chemistry , molecular dynamics , nmr spectra database , chemical shift , magic angle spinning , solid state nuclear magnetic resonance , crystallography , stereochemistry , spectral line , nuclear magnetic resonance spectroscopy , analytical chemistry (journal) , nuclear magnetic resonance , computational chemistry , physics , chromatography , astronomy
The global fold of human cannabinoid type 2 (CB 2 ) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state 13 C‐ and 15 N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly 13 C‐ and 15 N‐labeled CB 2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. 13 C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of C α , C β , and CO bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the 13 C chemical shift distribution of C α resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB 2 . Thus the shape of the C α band can be used as an indicator of CB 2 global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional 13 C α ( i ) 13 CO( i ) and 13 CO( i ) 15 NH( i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB 2 by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc.