Unnatural amino acid replacement in a yeast G protein-coupled receptor in its native environment

Biochemistry
Liyin HuangJeffrey M Becker

Abstract

Ste2p is the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha factor of Saccharomyces cerevisiae. This receptor-pheromone pair has been used extensively as a paradigm for investigating GPCR structure and function. Expression in yeast harboring a cognate tRNA/aminoacyl-tRNA synthetase pair specifically evolved to incorporate p-benzoyl- l-phenylalanine (Bpa) in response to the amber codon allowed the biosynthesis of Bpa-substituted Ste2p in its native cell. We replaced natural amino acid residues in Ste2p with Bpa by engineering amber TAG stop codons into STE2 encoded on a plasmid. Several of the expressed Bpa-substituted Ste2p receptors exhibited high-affinity ligand binding, and incorporation of Bpa into Ste2p influenced biological activity as measured by growth arrest of whole cells in response to alpha factor. We found that, at concentrations of 0.1-0.5 mM, a dipeptide containing Bpa could be used to enhance delivery of Bpa into the cell, while at 2 mM, both dipeptide and Bpa were equally effective. The application of a peptide delivery system for unnatural amino acids will extend the use of the unnatural amino acid replacement methodology to amino acids that are impermeable to yeast. Incorporation of ...Continue Reading

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Jan 20, 2009·Biochemistry·George K E UmanahJeffrey M Becker
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