posted on 2021-10-01, 15:05authored byXiaoyan Wang, Dongsheng Liu, Ling Shen, Fahui Li, Yongze Li, Lingyun Yang, Tiandan Xu, Houchao Tao, Deqiang Yao, Lijie Wu, Kunio Hirata, Laura M. Bohn, Alexandros Makriyannis, Xiaohong Liu, Tian Hua, Zhi-Jie Liu, Jiangyun Wang
Due
to the lack of genetically encoded probes for fluorine-19 nuclear
magnetic resonance spectroscopy (19F NMR), its utility
for probing eukaryotic membrane protein dynamics is limited. Here
we report an efficient method for the genetic incorporation of an
unnatural amino acid (UAA), 3′-trifluoromenthyl-phenylalanine
(mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression
System. The probe can be inserted at any environmentally sensitive
site, while causing minimal structural perturbation to the target
protein. Using 19F NMR and X-ray crystallography methods,
we discovered that the allosteric modulator Org27569 and agonists
synergistically stabilize a previously unrecognized pre-active state.
An allosteric modulation model is proposed to explain Org27569’s
distinct behavior. We demonstrate that our site-specific 19F NMR labeling method is a powerful tool in decoding the mechanism
of GPCR allosteric modulation. This new method should be broadly applicable
for uncovering conformational states for many important eukaryotic
membrane proteins.