Understanding the structure and function of proteins, the machines of life, underpins our knowledge of many biological processes. Several techniques are available to structurally interrogate proteins and their complexes, a number of which require the introduction of chemical labels into the protein(s) of interest. Continued expansion of the molecular “tool box” of labelling reagents and methods available to structural biologists is required to enable the study of more complex proteins and their interactions, including ones that may be targeted for therapeutic benefit.
Our recent contribution to this field has centred around the site-specific insertion of unnatural amino acids (UAAs) into proteins, using evolved orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs to genetically encode UAAs at chosen positions in response to the “amber” stop codon during in vivo protein synthesis.1 A diverse range of UAAs have been used to install photo-reactive groups, spin labels or paramagnetic lanthanides into proteins via such methodology. In combination with NMR spectroscopy,2 EPR spectroscopy3 and mass spectrometry, this is facilitating the generation of accurate atomic-resolution structural models of proteins, protein-ligand complexes and bio-macromolecular assemblies.
This presentation will discuss the synthesis and application of some of the UAAs and reactive labels prepared in our laboratory, and in so doing, highlight how stop codon reassignment technology can be applied to the structural analysis of a range of target proteins and complexes of biological/medicinal interest.