NMR characterization of recombinant transmembrane protein CB2 fragment CB2(180-233).
The expression of membrane proteins has been the bottleneck for their structural studies. Recently, we developed a method to obtain milligram quantities of isotope-labeled seven transmembrane G-protein coupled cannabinoid (CB) receptor fragment in E. coli. In order to verify this method and confirm the recombinant isotope-labeled CB2 fragment, 3D hetero-nuclear NMR techniques were used to analyze the structure of the fragment CB2(180-233) in DMSO-d6 solvent. The sequential assignments of TM5 and intra-cellular loop 3 were accomplished, which confirmed the experimental protocols of isotope-labeled recombinant protein expression, fusion protein cleavage, and membrane protein purification. The obtained structure also showed alpha-helix in the TM5 region, but it was interrupted by a disordered region (Gly204_ILe206). These results further revealed that our established approach is a promising method to express recombinant membrane proteins for their structural studies.
Zhao
J
J
Department of Pharmaceutical and Pharmacological Sciences, College of Pharmacy, University of Houston, TX 77204-5037, USA.
Zheng
H
H
Xie
X-Q
XQ
D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D043885:Receptor, Cannabinoid, CB2 / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry
D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D043885:Receptor, Cannabinoid, CB2; D011994:Recombinant Proteins