Horst Pick

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.

Visualizing odorant receptor trafficking in living cells down to the single-molecule level

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of ≈190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.

Characterization of an extended receptive ligand repertoire of the human olfactory receptor OR17-40 comprising structurally related compounds

Molecular properties of odorant compounds essential for activation of the human olfactory receptor hOR17‐40 were investigated using a collection of 23 variants of its cognate ligand helional. Coupling receptor activation to an optically detectable intracellular Ca2+ ion flux allowed dose‐dependent screening of different odorant molecules in human embryonic kidney (HEK)293 cells. We found an extended collection of activating ligands and provide first evidence for hOR17‐40‐specific antagonists. The C‐terminal fusion of enhanced green fluorescent protein to the hOR17‐40 retained full receptor function and permitted the selection of cells with defined receptor expression levels, which was an essential step for optimizing our screening protocol. Interestingly, cells with a low EGFP fluorescence intensity exhibited efficient hOR17‐40 cell surface targeting and odorant‐evoked signal transduction; in contrast, highly fluorescent cells displayed mainly incorrectly targeted, intracellular receptors. Fluorescence‐activated cell sorting was used to separate hOR17‐40‐expressing cells on the basis of their endogenous EGFP fluorescence intensity, thereby increasing the fraction of odorant‐responsive cells to up to 80% of the total cell number.

Large-scale production and study of a synthetic G protein-coupled receptor: Human olfactory receptor 17-4

Although understanding of the olfactory system has progressed at the level of downstream receptor signaling and the wiring of olfactory neurons, the system remains poorly understood at the molecular level of the receptors and their interaction with and recognition of odorant ligands. The structure and functional mechanisms of these receptors still remain a tantalizing enigma, because numerous previous attempts at the large-scale production of functional olfactory receptors (ORs) have not been successful to date. To investigate the elusive biochemistry and molecular mechanisms of olfaction, we have developed a mammalian expression system for the large-scale production and purification of a functional OR protein in milligram quantities. Here, we report the study of human OR17-4 (hOR17-4) purified from a HEK293S tetracycline-inducible system. Scale-up of production yield was achieved through suspension culture in a bioreactor, which enabled the preparation of >10 mg of monomeric hOR17-4 receptor after immunoaffinity and size exclusion chromatography, with expression yields reaching 3 mg/L of culture medium. Several key post-translational modifications were identified using MS, and CD spectroscopy showed the receptor to be ≈50% α-helix, similar to other recently determined G protein-coupled receptor structures. Detergent-solubilized hOR17-4 specifically bound its known activating odorants lilial and floralozone in vitro, as measured by surface plasmon resonance. The hOR17-4 also recognized specific odorants in heterologous cells as determined by calcium ion mobilization. Our system is feasible for the production of large quantities of OR necessary for structural and functional analyses and research into OR biosensor devices.

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.

Sequential stages in the life cycle of the ionotropic 5-HT3 receptor (5-HT3R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT3R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT3R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT3 R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT3 receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 μm/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT3 receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.

Monitoring Tyrosinase Expression in Non-metastatic and Metastatic Melanoma Tissues by Scanning Electrochemical Microscopy

Although tremendous progress has been made in the diagnosis of melanoma, the identification of different stages of malignancy in a reliable way remains challenging. Current strategies rely on optical monitoring of the concentration and spatial distribution of specific biomarkers. State-of-the-art optical methods can be affected by background-color interference and autofluorescence. We overcame these shortcomings by employing scanning electrochemical microscopy (SECM) to map the prognostic indicator tyrosinase (TyR) in non-metastatic and metastatic melanoma tissues by using soft-stylus microelectrodes. Electrochemical readout of the TyR distribution was enabled by adapting an immunochemical method. SECM can overcome the limitations of optical methods and opens unprecedented possibilities for improved diagnosis and understanding of the spatial distribution of TyR in different melanoma stages.

Ligand binding transmits conformational changes across the membrane-spanning region to the intracellular side of the 5-HT3 serotonin receptor

Conformational changes of channel activation: Five enhanced green fluorescent protein (EGFP) mols. (green cylinders) were integrated into the intracellular part of the homopentameric ionotropic 5-HT3 receptor. This allowed the detection of extracellular binding of fluorescent ligands (.bul.) to EGFP by FRET, and also enabled the quantification of agonist-induced conformational changes in the intracellular region of the receptor by homo-FRET between EGFPs. The approach opens novel ways for probing receptor activation and functional screening of therapeutic compds. [on SciFinder (R)]

Recombinant Expression and Functional Characterization of Mouse Olfactory Receptor mOR256-17 in Mammalian Cells

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system in recognizing and discriminating a nearly unlimited number of structurally diverse odorous molecules. The molecular basis of OR-mediated signal detection and transduction is poorly understood. This is due to difficulties in functional expression of ORs in high yields, preventing structural and biophysical studies at the level of the receptor protein. Here we report on recombinant expression of mouse receptor mOR256-17 yielding 10(6) ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression, we employed different fluorescent probes. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and post-translational fluorescence labeling of a 12-amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. Our dual-color labeling approach is generally applicable to quantification of membrane proteins for mammalian cell-based expression. By screening a large odorant compound library, we discovered a selective spectrum of potent mOR256-17-specific agonists essential for probing the receptor function for future scaled-up productions.

Engineering chromatin states: Chemical and synthetic biology approaches to investigate histone modification function ☆

Patterns of histone post-translational modifications (PTMs) and DNA modifications establish a landscape of chromatin states with regulatory impact on gene expression, cell differentiation and development. These diverse modifications are read out by effector protein complexes, which ultimately determine their functional outcome by modulating the activity state of underlying genes. From genome-wide studies employing high-throughput ChIP-Seq methods as well as proteomic mass spectrometry studies, a large number of PTMs are known and their coexistence patterns and associations with genomic regions have been mapped in a large number of different cell types. Conversely, the molecular interplay between chromatin effector proteins and modified chromatin regions as well as their resulting biological output is less well understood on a molecular level. Within the last decade a host of chemical approaches has been developed with the goal to produce synthetic chromatin with a defined arrangement of PTMs. These methods now permit systematic functional studies of individual histone and DNA modifications, and additionally provide a discovery platform to identify further interacting nuclear proteins. Complementary chemical- and synthetic-biology methods have emerged to directly observe and modulate the modification landscape in living cells and to readily probe the effect of altered PTM patterns on biological processes. Herein, we review current methodologies allowing chemical and synthetic biological engineering of distinct chromatin states in vitro and in vivo with the aim of obtaining a molecular understanding of histone and DNA modification function. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.