Abstract
Site-selective protein labeling is an indispensible approach in the currently intense chemical biology research area. Studies involving site-selective protein labeling span from the protein dynamic analysis in vitro to the proteinprotein interaction investigation in living cells. In the past decade, multiple methods have been introduced to achieve site-selective protein labeling. These include genetic fusion of green fluorescent protein and its derivatives, selective chemical labeling of proteins with fusion tags, and site-specific modification of noncanonical amino acids that are genetically encoded. Using evolved orthogonal aminoacyl-tRNA synthetase-nonsense suppressor tRNA pairs, noncanonical amino acids with bioorthogonal functional groups such as azide, alkyne, tetrazine, alkene, keto, phenylhalide, etc. have been genetically incorporated into proteins in E. coli, yeast, and mammalian cells. Genetic encoding of these noncanonical amino acids enables multiple ways for site-selective protein labeling both in vitro and in vivo, allowing diverse strategies to interrogate protein functions. This review intends to provide a brief introduction to the genetic noncanonical amino acid incorporation technique and recent progresses in applying this technique to achieve site-selective protein labeling.
Highlights
Protein labeling with fluorescent molecules that allows sensing and visualization of protein dynamics, localization, protein-ligand interactions, and protein-protein interactions, is an invaluable tool to understand protein functions in living cells
green fluorescent protein (GFP) variants have proved to be extremely useful for both in vitro and in vivo studies of protein functions, their utility is still limited because the molecular sizes of GFP variants (~27kDa) are large enough to potentially interfere with the structure and function of proteins to which they are fused and their spectral and structural characterization are interdependent [68]
To genetically encode a keto functional group, Schultz and coworkers evolved several MjTyrRS variants for specific incorporation of a NAA 4 shown in Figure 2 into proteins in E. coli [62]. 4 has been genetically encoded in yeast and mammalian cells using evolved tyrosyl-tRNA synthetase (EcTyrRS)-amber suppressing tRNACTyUrA pairs that were derived from E. coli [53,54]
Summary
Protein labeling with fluorescent molecules that allows sensing and visualization of protein dynamics, localization, protein-ligand interactions, and protein-protein interactions, is an invaluable tool to understand protein functions in living cells. An ideal chemical labeling approach that can achieve comparable simplicity and efficiency of the GFP labeling technique is to directly incorporate fluorescent NAAs into proteins. In order to undergo clicktype protein-labeling reactions, bioorthogonal functional groups that do not exist in the biological system can be introduced into proteins followed by selective reactions with fluorophore-containing dyes.
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