A general method for the covalent labeling of fusion proteins with small molecules in vivo.
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.
Keppler
Antje
A
Institute of Molecular and Biological Chemistry, Swiss Federal Institute of Technology Lausanne (EPFL), CH-1015, Lausanne, Switzerland.
Gendreizig
Susanne
S
Gronemeyer
Thomas
T
Pick
Horst
H
Vogel
Horst
H
Johnsson
Kai
K
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D006224:Cricetinae; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D015003:Yeasts / Q000737:chemistry / Q000166:cytology / Q000378:metabolism
D008024:Ligands; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019853:O(6)-Methylguanine-DNA Methyltransferase; D019793:Fluorescein