Antje Keppler

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.

Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum

O6-alkylguanine-DNA alkyltransferase (AGT) fusion proteins can be specifically and covalently labeled with fluorescent O6-benzylguanine (O6-BG) derivatives for multicolor live cell imaging approaches. Here, we characterize several new BG fluorophores suitable for in vivo AGT labeling that display fluorescence emission maxima covering the visible spectrum from 472 to 673 nm, thereby extending the spectral limits set by fluorescent proteins. We show that the photostability of the cell-permeable dyes BG Rhodamine Green (BG505) and CP tetramethylrhodamine (CP-TMR) is in the range of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP), and that BG diethylaminomethyl coumarin (BGDEAC), a derivative of coumarin, is even more stable than enhanced cyan fluorescent protein (ECFP). Due to the increasing number of new BG derivatives with interesting fluorescence properties, such as far-red emission, fluorescence labeling of AGT fusion proteins is becoming a versatile alternative to existing live cell imaging approaches.

Chromophore-assisted laser inactivation of alpha- and gamma-tubulin SNAP-tag fusion proteins inside living cells.

Chromophore-assisted laser inactivation (CALI) can help to unravel localized activities of target proteins at defined times and locations within living cells. Covalent SNAP-tag labeling of fusion proteins with fluorophores such as fluorescein is a fast and highly specific tool to attach the photosensitizer to its target protein in vivo for selective inactivation of the fusion protein. Here, we demonstrate the effectiveness and specificity of SNAP-tag-based CALI by acute inactivation of alpha-tubulin and gamma-tubulin SNAP-tag fusions during live imaging assays of cell division. Singlet oxygen is confirmed as the reactive oxygen species that leads to loss of fusion protein function. The major advantage of SNAP-tag CALI is the ease, reliability, and high flexibility in labeling: the genetically encoded protein tag can be covalently labeled with various dyes matching the experimental requirements. This makes SNAP-tag CALI a very useful tool for rapid inactivation of tagged proteins in living cells.

Synthesis and Applications of Chemical Probes for Human O6‐Alkylguanine‐DNA Alkyltransferase

The design and synthesis of chem. probes to study and to elucidate complex biol. problems is becoming an increasingly important field in chem. We are interested in the repair of O6-alkylated guanines in DNA, a DNA lesion that results from alkylation by S-adenosylmethionine or exogenous toxins and which has been shown to be highly mutagenic and carcinogenic. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT; EC 2.1.1.63) reverses this alkylation by transferring the alkyl group to a reactive cysteine residue in the protein, leading to repaired DNA and an irreversibly alkylated protein. The expression level of human AGT (hAGT) in tumor cells is also crucial for their sensitivity to chemotherapeutic agents that alkylate DNA, such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide. Consequently, hAGT has become a target in cancer chemotherapy, as its inhibition would increase the efficiency of currently used DNA-alkylating drugs. A simple and reliable assay to measure the activity of hAGT in cell exts. would be of great importance for research on the role of hAGT in the chemotherapy of tumors, as currently used assays rely on radioactively labeled substrates and a subsequent HPLC sepn. We describe here the synthesis of oligonucleotides contg. O6-alkylated guanine derivs. of the type 1 and 2 that serve as affinity labels for hAGT and their use in a highly specific assay for this alkyltransferase. In addn., we introduce a novel system for the directed mol. evolution of hAGT, which relies on the display of active hAGT on phage l and on oligonucleotides contg. O6-alkylated guanine derivs. of the type 1 and 2. [on SciFinder (R)]

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)]