Expanding the chemistry of fluorescent protein biosensors through genetic incorporation of unnatural amino acids

Signal Transduction: From the Atomic Age to the Post-Genomic Era

“…For example, unnatural amino acid (UAA) technology (Liu and Schultz 2010; Chin 2014) offers another route for genetically encoding a protein with a site-specific fluorescent tag. By extending the genetic code and making the necessary changes to the translation machinery, one can incorporate at any specified position in a protein a 21st amino acid (either a UAA with a fluorescent side chain [Kang et al 2013; Niu and Guo 2013] or a UAA whose side chain can be readily coupled to a cell-permeable fluorophore [Borrmann et al 2012], or even two different UAAs at distinct sites in the same protein [Xiao et al 2013a]). Together, all of these methods provide a researcher with a broad palette for fluorescent labeling, permitting simultaneous interrogation of the locations of and/or conformational changes in multiple signaling proteins in the same cell.…”

Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle

“…18,25 SfGFP 26 is an intrinsically fluorescent protein derived from the original Aequorea victoria GFP. 27 Autofluorescent proteins such as GFP have proved to be an excellent model system for investigating and understanding the effect of ncAA incorporation on protein function (for examples see reviews 2,28 ); structural studies are crucial for understanding the molecular basis of action of ncAA engineering but are still relatively rare, 10,16,29–33 and even less so for ones that actively modulate function. Here we provide a timely structural insight and comparison concerning fluorescence switching on either click addition or irradiation of sfGFP containing the H148azF mutation (sfGFP 148azF ).…”

Site-specific incorporation of a fluorescent terphenyl unnatural amino acid

“…[4][5][6][7][8][9][10][11] Specifically, modifying proteins with fluorescent probes provides the ability to examine structure and function of proteins as well as visualize their cellular location via fluorescence spectroscopy. 12 Numerous fluorescent probes have been utilized within a protein context to further advance our understanding of proteins. Different fluorophores possess unique spectral properties for different applications, and thus the generation of multiple fluorophore labels is advantageous, and allows for adaptation to the needs of the experiment.…”