Unnatural amino acid replacement in a yeast G protein-coupled receptor in its native environment.
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 Bpa into Ste2p was verified by mass spectrometric analysis, and two Bpa-Ste2p mutants were able to selectively capture alpha factor into the ligand-binding site after photoactivation. To our knowledge, this is the first experimental evidence documenting an unnatural amino acid replacement in a GPCR expressed in its native environment and the use of a mutated receptor to photocapture a peptide ligand.
Huang
Li-Yin
LY
Department of Microbiology, UniVersity of Tennessee, KnoxVille, Tennessee 37996, USA.
Umanah
George
G
Hauser
Melinda
M
Son
Cagdas
C
Arshava
Boris
B
Naider
Fred
F
Becker
Jeffrey M
JM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008024:Ligands; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000596:Amino Acids; D008024:Ligands; D043562:Receptors, G-Protein-Coupled