Chemical labeling of protein in living cells.

Fluorescence imaging of fluorophore-labeled proteins opens a window into cellular protein biochemistry and enables direct visualization of protein dynamics, localization, and interactions in single living cells. Green fluorescent protein (GFP) and its color variants are popular fluorescent tags because they can be genetically fused to any protein of interest with great specificity. 2] However, their relatively large size (~238 amino acids) can potentially alter the structure and/or function of the host proteins, such as mislocalization or misexpression. Alternatively, chemoselective labeling of a protein in the context of the native cellular environment with small-molecule fluorescent probes could offer an exciting opportunity to expand the utility of in vivo protein imaging. Several smallmolecule-based chemical labeling methods have been developed to address this great challenge. A commonly used chemical-labeling method takes advantage of the specific interaction between a receptor protein and its small-molecule ligand. Just like GFP, the receptor protein is genetically fused to the protein of interest, and the ligand is synthetically conjugated to a fluorophore (Scheme 1A). The binding of the ligand to the receptor protein directs the fluorophore label to the fusion protein. In this way, any fluorophore with desirable optical properties can, in principle, be introduced to the fusion protein. An incopmlete list of receptor proteins includes E. coli dihydrofolate reductase (DHFR), 7] FK506-binding protein (FKBP12), a mutant of human O-alkylguanine-DNA transferase (hAGT), 10] a mutated prokaryotic dehalogenase (Halo ag< protein), small dye-binding peptides (in this case, the ligand itself is the fluorescent tag), and lanthanidebinding tags. Depending on the nature of the interaction between the receptor protein and the ligand, the labeling can be covalent (e.g. , in the case of hAGT and HaloTag protein) or noncovalent (e.g. , DHFR, FKBP12). The fusion protein may be located intracellularly or at the membrane. This method allows the incorporation of a variety of small-molecule fluorophores with diverse spectral properties into the fusion protein, particularly fluorophores with infrared or near-infrared emission, which are not available with GFP mutants. Tags for other imaging modalities, such as MRI, may be incorporated as well with this method. However, this labeling flexibility comes with several drawbacks. The size of the receptor proteins varies, but is often comparable to that of GFPs. The fluorescent emission of the tag is not modulated by binding, and a washing procedure is generally required to remove unbound ligands and minimize the background. If an endogenous equivalent of the receptor protein [a] Dr. A. Dragulescu-Andrasi, Prof. J. Rao Molecular Imaging Program at Stanford Department of Radiology & Bio-X Program Cancer Biology Program Stanford University School of Medicine 1201 Welch Road Stanford, CA 94305-5484 (USA) Fax: (+1)650-736-7925 E-mail : jrao@stanford.edu Scheme 1. Chemical methods for in vivo protein labeling. A) Receptor-protein-based method. The fluorescent probe is linked to an affinity ligand that specifically binds to its receptor. B) Enzyme-mediated ACHTUNGTRENNUNGligation. The probe is linked to a substrate that can be ligated onto a peptide tag genetically fused to the host protein by a “third-party” enzyme. C) A fluorogenic probe binds to a peptide tag–protein fusion, which activates its fluorescence.