A general method for the covalent labeling of fusion proteins with small molecules in vivo

Abstract

Characterizing the movement, interactions, and chemical microenvironment of a protein inside the living cell is crucial to a detailed understanding of its function. Most strategies aimed at realizing this objective are based on genetically fusing the protein of interest to a reporter protein that monitors changes in the environment of the coupled protein. Examples include fusions with fluorescent proteins, the yeast two-hybrid system, and split ubiquitin. However, these techniques have various limitations, and considerable effort is being devoted to specific labeling of proteins in vivo with small synthetic molecules capable of probing and modulating their function. These approaches are currently based on the noncovalent binding of a small molecule to a protein, the formation of stable complexes between biarsenical compounds and peptides containing cysteines, or the use of biotin acceptor domains. Here we describe a general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalent labeling of proteins and that may open up new ways of studying proteins in living cells.

Analysis of Protein Turnover by Quantitative SNAP‐Based Pulse‐Chase Imaging

“…It acts as a suicide enzyme that catalyzes a covalent binding reaction between itself and the alkyl group that is removed from guanines, thereby restoring DNA integrity but inactivating its own catalytic activity (Pegg, 2000). SNAP, the modified form of hATG, has lost its affinity to DNA but efficiently reacts with soluble O 6 -benzylguanine (BG), of which the benzyl moiety is readily transferred to the SNAP protein ( Figure 1, Juillerat et al, 2003;Keppler et al, 2003). The benzyl rings in BG can be coupled to a large variety of molecules (Keppler et al, , 2004(Keppler et al, , 2006) that include fluorescent moieties as well as non-fluorescent ones (a selection of SNAP substrates is presented in Table 2).…”

“…Here we discuss SNAP-based pulse-chase imaging, a powerful method to track protein dynamics with distinct advantages over traditional methods to assess protein dynamics. SNAP is a suicide enzyme protein fusion tag that catalyzes its own covalent binding to the cell permeable molecule benzylguanine (BG), and (fluorescent) derivatives thereof ( Figure 1; Damoiseaux et al, 2001;Keppler et al, 2003Keppler et al, , 2004. Fusion of SNAP to a protein of interest allows this protein to be (fluorescently) labeled at will in living cells.…”

Chemical tags: Applications in live cell fluorescence imaging

“…The hAGT protein transfers alkyl groups from the O 6 -position of the guanine in a one turnover self-modification reaction to a cysteine residue in its active site [4]. Johnsson and colleagues showed that modified O 6 -benzyl guanine substrates carrying fluorophores in the para position of the benzyl group result in labeling of hAGT at the active cysteine residue [5]. By mutational analysis they identified a hAGT mutant that catalyzed the self-labeling reaction 50 times faster than the wild type hAGT, thus eliminating the need for hAGT deficient cell lines [6][7].…”

Metal-Based Optical Probes for Live Cell Imaging of Nitroxyl (HNO)

“…Strategies to label particular cellular organelles include genetically encoded fusion protein tags, [40][41][42] synthetic peptides, [43][44][45][46] and smallmolecule vectors [47][48][49] to deliver HNO sensors to programmed locations within the cell.…”