Thomas Gronemeyer

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.

Directed evolution of O6-alkylguanine-DNA alkyltransferase for efficient labeling of fusion proteins with small molecules in vivo

We report here the generation of mutants of the human O(6)-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter molecules. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O(6)-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O(6)-benzylguanine derivatives, the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small molecules and therefore should become important tools for studying protein function in living cells. In addition to various applications in proteomics, the selected mutants also yield insight into the interaction of the DNA repair protein hAGT with its inhibitor O(6)-benzylguanine.

Covalent labeling of fusion proteins with chemical probes in living cells

A general method for the specific and covalent labeling of fusion proteins in vivo is described. The approach is based on the unusual mechanism of the human DNA repair protein O6-alkylguanine-DNA alkyl-transferase, which irreversibly transfers an alkyl group from O6-alkylguanine-DNA to one of its cysteine residues. Labeling is possible in bacterial as well as eukaryotic cells and is independent of the nature of the label, thereby opening up new ways to study proteins in vivo. [on SciFinder (R)]