Incorporation of the Unnatural Amino Acid p-benzoyl-L-phenylalanine (Bpa) into a G Protein-coupled Receptor in its Native Context
Ligand binding initiates a change in the conformation of G protein-coupled receptors (GPCRs) resulting in activation of the G protein-mediated signal transduction cascade . We are studying a novel approach to elucidate the dynamics of GPCR structure by the co-translational introduction of unnatural amino acids (UAAs) into the receptor.
UAAs can be synthesized to contain a variety of chemical moieties for use as photoaffinity labels, fluorescent labels, or spectroscopic probes. Orthogonal tRNA/ aminoacyl-tRNA synthetase pairs evolved and expressed in the target cell have been used to incorporate UAAs into heterologously expressed protein in living cells . The mutated tRNA, charged with its UAA, recognizes the amber TAG stop codon and incorporates the non-natural amino acid into the nascent polypeptide chain (See Figure below). UAAR has been widely used in the heterologous Xenopusoocyte expression system to insert UAAs into a variety of receptors and channel...
KeywordsUnnatural Amino Acid Aminoacyl tRNA Synthetase Amino Acid Analog Nascent Polypeptide Chain Eukaryotic Host Cell
This work was supported by grants NIH GM-22086(FN) and NIH GM-22087(JMB).