UAAM:  Unnatural Amino Acid Mutagenesis

Summary.  The Sakmar laboratory has pioneered novel approaches to introduce non-perturbing chemical or spectroscopic labels into low abundance cell surface receptors such as heptahelical G protein-coupled receptor (GPCRs).  Recently, in an interdisciplinary collaboration with Prof. U. L. RajBhandary at M.I.T., the Sakmar Laboratory developed an amber codon suppression strategy suitable for unnatural amino acid (UAA) mutagenesis of proteins expressed in mammalian cells.  Members of the Sakmar Laboratory engineered suitable suppressor tRNAs and orthogonal amino acyl-tRNA synthetases that suppress the termination of protein synthesis at an amber codon mutation in a gene of interest and introduce a UAA.  The UAA mutagenesis method was used to introduce p-azido-L-phenylalanine (azF), p-acetyl-L-phenylalanine (acF) or p-benzoyl-L-phenylalanine (bzF) into rhodopsin or CCR5 at high yield. 

How Does UAAM Work?  
In site-directed UAAM, engineered amino acyl-tRNA synthetase constructs are designed to bind specific UAAs and transfer them to engineered suppressor tRNAs. Together the orthogonal pairs of engineered aminoacyl-tRNA synthetase and tRNA cause the incorporation, at the site of an amber codon mutation, of a particular UAA added to the culture medium.  In approximately two decades since the first demonstration of site-directed UAAM, various amino acid analogues with novel chemical or physical, properties have been incorporated into proteins expressed in E. coli or using in vitro protein translation systems.  However, the methods developed for use in E. coli do not work in mammalian cells.  To adapt UAAM approach for use in mammalian cells, the Sakmar Laboratory employed a cell-based luciferase screen to select for efficient amber codon suppression by testing a variety of engineered suppressor tRNA constructs and engineered amino acyl-tRNA synthetases.  The mammalian expression of a particular engineered Bacillus stearathermophilus tyrosine-tRNA was optimized and paired with an engineered E. coli tyrosine amino-acyl tRNA synthetase.

As a proof-of-concept, the Sakmar Laboratory incorporated three tyrosine analogues at high efficiency in two different GPCRs – rhodopsin (the photoreceptor for the dim-light vision) and CCR5 (a chemokine receptor that has been identified to be the co-receptor for the HIV viral entry into T-cells).  The strategy was to target specific sites on the receptor while preserving its function.  The expression yields of functional receptors were evaluated by spectroscopic quantification of rhodopsin or calcium flux functional assay of CCR5.  The results demonstrated the general utility of the novel UAA mutagenesis technology.  High efficiency UAA mutagenesis of membrane receptors in mammalian cells can be used to create reactive tags tailored for the site-specific post-translational labeling application.

Potential Impact of the UAAM Technology. 
Members of the Sakmar Laboratory have optimized and validated orthogonal tRNA-amino acyl tRNA synthetase pairs to incorporate p-azido-L-phenylalanine (azF), p-acetyl-L-phenylalanine (acF) or p-benzoyl-L-phenylalanine (bzF) into heterologously-expressed proteins in mammalian cells.  These particular UAAs were chosen because they contain functional groups – keto for acF and bzF, and azido (N3) for azF – not normally present on cell proteins.  These functional groups, therefore, provide a specific reactive handle to label proteins with fluorophores or other informative probes.  In addition, the benzyol and azido groups are photoreactive and provide the potential to carry out photochemical cross-linking experiments.  Finally, the azido group has a unique infrared vibrational frequency useful for Fourier-transform infrared spectroscopy. 
To label proteins post-translationally, members of the Sakmar Laboratory are using the Staudinger-Bertozzi ligation reaction and the [3+2] cyclo-addition, “click” chemistry, reaction.  Preliminary results are encouraging even under physiological reactions conditions, which do not denature or disrupt the normal structure or function of the target protein.  Using the UAAM procedure and post-translational labeling, possibly in combination with other methods, it should be possible to introduce multiple labels on one protein or different labels on different proteins.  Thus, donor-acceptor fluorescence resonance energy transfer (FRET) pairs can be introduced to monitor intra- or intermolecular dynamic processes.