Rapid bioorthogonal labeling of proteins
By Clay Clark, @biochemprof
There are a number of current methods for labeling proteins for imaging either in vitro and/or in live cells and organisms, including fusions with fluorescent proteins, dyes, tags (such as SNAP, HALO, CLIP), ligases, spin-labels, or unnatural amino acids. Some of the methods are summarized in the figure from Chen & Ting, which shows several approaches to labeling proteins with small molecules, either through fusion proteins (A), chemical or enzymatic labeling of a protein tag (B,C) or site-specific labeling using genetically encoded amber mutations (D).
In recent years, several chemical probes have been developed that allow the incorporation of reactive tags into proteins. The tags can then be modified within the complex mixture of the cellular milieu, providing a powerful technique to examine protein structure and function as well as interaction networks in native conditions (see review of the tags by Best).
While some of the tags allow rapid labeling and have been used in numerous cell biological studies, many of the methods result in the addition of extra amino acids to the protein, which may affect the protein structure or function. In addition, some of the chemical probes have slow reaction times, which limits their use.
In a recent paper by Chin and colleagues, published in Nature Chemistry, the authors used a bioorthogonal approach coupled to rapid reaction chemistry to label proteins. In this approach, a genetically encoded amber suppressor aminoacyl tRNA synthetase & tRNACUA pair from Methanosarcina species was used to site-specifically label proteins. The pyrrolysyl-tRNA syntheses from M. barkeri and M. mazei incorporate pyrrolysine into an amber stop codon introduced in the gene of interest. In this study, the authors modified lysine to contain norbornene (Nε-tert-butyloxycarbonyl-L-lysine) or other groups.
The authors used the PylRS/tRNACUA pair to incorporate the norbornene-containing amino acid into model proteins, GFP and myoglobin, recombinantly expressed in E. coli. An advantage of the norbornene probe is that the protein can be labelled rapidly and selectively with tetrazine-based probes and monitored by fluorescence emission. The tetrazine-based probes have low background because they are “turn-on” fluorescent probes; the fluorescence is high only after reaction with the norbornene label on the protein. Experiments with the model proteins show that the probe reacts efficiently and specifically with the norbornene-labeled proteins.
The authors then examined labeling of mammalian cell (HEK293) surfaces by introducing an amber codon into the extracellular region of an EGFR that was also fused to GFP, such that the GFP was intracellular. The efficiency and specificity of labeling could then be tested by examining GFP fluorescence as well as protein labeled with a tetrazine-TAMRA conjugate, which fluoresces red.
Overall, Chin and colleagues show the site-specific introduction of fluorescent probes into proteins using the genetically encoded incorporation of a norbornene-containing amino acid. The methods were shown to be efficient in both E. coli and mammalian cells and provide a new and efficient tool to examine proteins in their native environment.
Lang, K., Davis, L., Torres-Kolbus, J., Chou, C., Deiters, A., & Chin, J. (2012). Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction Nature Chemistry DOI: 10.1038/NCHEM.1250
Chen, I., & Ting, A. (2005). Site-specific labeling of proteins with small molecules in live cells Current Opinion in Biotechnology, 16 (1), 35-40 DOI: 10.1016/j.copbio.2004.12.003
Best, M. (2009). Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules Biochemistry, 48 (28), 6571-6584 DOI: 10.1021/bi9007726