Noncanonical Amino Acid Tools and Their Application to Membrane Protein Studies

Methods rooted in chemical biology have contributed significantly to studies of integral membrane proteins. One recent key approach has been the application of genetic code expansion (GCE), which enables the site-specific incorporation of noncanonical amino acids (ncAAs) with defined chemical properties into proteins. Efficient GCE is challenging, especially for membrane proteins, which have specialized biogenesis and cell trafficking machinery and tend to be expressed at low levels in cell membranes. Many eukaryotic membrane proteins cannot be expressed functionally in E. coli and are most effectively studied in mammalian cell culture systems. Recent advances have facilitated broader applications of GCE for studies of membrane proteins. First, AARS/tRNA pairs have been engineered to function efficiently in mammalian cells. Second, bioorthogonal chemical reactions, including cell-friendly copper-free “click” chemistry, have enabled linkage of small-molecule probes such as fluorophores to membrane proteins in live cells. Finally, in concert with advances in GCE methodology, the variety of available ncAAs has increased dramatically, thus enabling the investigation of protein structure and dynamics by multidisciplinary biochemical and biophysical approaches. These developments are reviewed in the historical framework of the development of GCE technology with a focus on applications to studies of membrane proteins.