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.

A general approach for the sequential labeling of fusion proteins of O 6 -alkylguanine-DNA alkyltransferase (AGT) with different fluorophores in mammalian cells is presented. AGT fusion proteins with different localizations in the cell can be labeled specifically with different fluorophores, and the fluorescence labeling can be used for applications such as multicolor analysis of dynamic processes and fluorescence resonance energy transfer measurements. The facile access to a variety of different AGT substrates as well as the specificity of the labeling reaction should make the approach an important tool to study protein function in live cells.A dvances in fluorescence microscopy and the isolation and engineering of different autofluorescent proteins have revolutionized the way protein function is studied in the living cell (1-3). Beyond localization studies, fusion proteins of autofluorescent proteins can be used to study dynamic processes, follow conformational changes, and detect protein-protein interactions (1, 2, 4). Despite this enormous range of applications for autofluorescent fusion proteins, there are two reoccurring limitations of the approach. The first limitation is the restriction to the fluorophores of the existing autofluorescent proteins. Properties such as emission and excitation wavelength, extinction coefficient, and photostability are characteristic properties of each autofluorescent protein that do not necessarily match the requirements of the envisioned application. The second limitation is caused by the fusion of the autofluorescent protein to the protein of interest. In general, autofluorescent proteins possess a molecular mass of Ͼ25 kDa, and certain autofluorescent proteins furthermore form oligomers, raising the question of to what extent the creation of a fusion protein affects the function of the protein of interest (1, 3).Alternative approaches to make proteins amenable to fluorescence studies in live cells include introduction of chemically labeled proteins through invasive techniques such as microinjection, site-specific incorporation of unnatural fluorescent amino acids, and the selective labeling of fusion proteins (5-10). An example of the latter approach is the so-called tetracysteine tag, which reacts with biarsenical fluorophores to form stable and highly fluorescent complexes (7,8). We recently developed an approach that allows for the labeling of fusion proteins of human O 6 -alkylguanine-DNA alkyltransferase (AGT) with synthetic molecules (9). The labeling is based on the irreversible and specific reaction of AGT with O 6 -benzylguanine (BG) derivatives, leading to the transfer of the synthetic probe to a reactive cysteine residue (Fig. 1). Wild-type human AGT is a monomeric protein of 207 aa, the 30 C-terminal residues of which can be deleted without affecting the activity against BG, making it slightly smaller than autofluorescent proteins (11). Importantly, the rate of the reaction of AGT fusion proteins with BG derivatives is independent of the nature of the label, o...

The specific and covalent labeling of fusion proteins with synthetic molecules opens up new ways to study protein function in the living cell. Here we present a novel method that allows for the specific and exclusive extracellular labeling of proteins on the surfaces of live cells with a large variety of synthetic molecules including fluorophores, protein ligands, or quantum dots. The approach is based on the specific labeling of fusion proteins of acyl carrier protein with synthetic molecules through post-translational modification catalyzed by phosphopantetheine transferase. The specificity and versatility of the labeling should allow it to become an important tool for studying and manipulating cell surface proteins and for complementing existing approaches in cell surface engineering.

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.

Understanding cellular signaling mediated by cell surface receptors is key to modern biomedical research and drug development. The discovery of a growing number of potential molecular targets and therapeutic compounds requires downscaling and accelerated functional screening. Receptor-mediated cellular responses are typically investigated on single cells or cell populations. Here, we show how to monitor cellular signaling reactions at a yet unreached miniaturization level. On the basis of our observations, cytochalasin induces mammalian cells to extrude from their plasma membrane submicrometer-sized native vesicles. They comprise functional cell surface receptors correctly exposing their extracellular ligand binding sites on the outer vesicle surface and retaining cytosolic proteins in the vesicle interior. As a prototypical example, ligand binding to the ionotropic 5-HT(3) receptor and subsequent transmembrane Ca(2+) signaling were monitored in single attoliter vesicles. Thus, native vesicles are the smallest autonomous containers capable of performing cellular signaling reactions under physiological conditions. Because a single cell delivers about 50 native vesicles, which can be isolated and addressed as individuals, our concept allows multiple functional analyses of individual cells having a limited availability and opens new vistas for miniaturized bioanalytics.