Site-specific, orthogonal labeling of proteins in intact cells with two small biarsenical fluorophores. 2010

Alexander Zürn, and Christoph Klenk, and Ulrike Zabel, and Susanne Reiner, and Martin J Lohse, and Carsten Hoffmann
Institute of Pharmacology and Toxicology, Rudolf Virchow Center University of Wurzburg, Versbacher Strasse 9, 97078 Wurzburg, Germany.

The fusion of fluorescent proteins to proteins of interest has greatly advanced fluorescence microscopy, but is often limited by their large size. Here, we report site-specific, orthogonal labeling of two cellular proteins in intact cells with two small fluorescent dyes: fluorescein arsenical hairpin binder, FlAsH, and its red analogue, ReAsH, which bind to tetracysteine motifs. Development of a sequential labeling method to two different motifs, CCPGCC and FLNCCPGCCMEP, allowed site-specific labeling with FlAsH and ReAsH, respectively. Using the cell surface receptor for parathyroid hormone and its cytosolic binding protein, beta-arrestin2, we show their selective visualization in intact cells and analyze their interaction by colocalization and fluorescence resonance energy transfer (FRET). We propose that this method may be widely applied to label intracellular proteins and to study their interactions in intact cells with minimal disturbance of their function.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011485 Protein Binding The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments. Plasma Protein Binding Capacity,Binding, Protein
D011506 Proteins Linear POLYPEPTIDES that are synthesized on RIBOSOMES and may be further modified, crosslinked, cleaved, or assembled into complex proteins with several subunits. The specific sequence of AMINO ACIDS determines the shape the polypeptide will take, during PROTEIN FOLDING, and the function of the protein. Gene Products, Protein,Gene Proteins,Protein,Protein Gene Products,Proteins, Gene
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D005456 Fluorescent Dyes Chemicals that emit light after excitation by light. The wave length of the emitted light is usually longer than that of the incident light. Fluorochromes are substances that cause fluorescence in other substances, i.e., dyes used to mark or label other compounds with fluorescent tags. Flourescent Agent,Fluorescent Dye,Fluorescent Probe,Fluorescent Probes,Fluorochrome,Fluorochromes,Fluorogenic Substrates,Fluorescence Agents,Fluorescent Agents,Fluorogenic Substrate,Agents, Fluorescence,Agents, Fluorescent,Dyes, Fluorescent,Probes, Fluorescent,Substrates, Fluorogenic
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000071557 beta-Arrestins Non-visual system arrestins that negatively regulate G-PROTEIN-COUPLED RECEPTORS (GPCRs) and may also function independently of GPCR signaling. They bind and recruit many different signaling factors, including MITOGEN-ACTIVATED PROTEIN KINASES; SRC-FAMILY-KINASES; and FILAMIN to GPCRs and may recognize different phosphorylation states of the receptors to determine the specificity of the cellular response to signaling. beta-Arrestin,beta Arrestin,beta Arrestins
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D044168 Receptor, Parathyroid Hormone, Type 1 A parathyroid hormone receptor subtype that recognizes both PARATHYROID HORMONE and PARATHYROID HORMONE-RELATED PROTEIN. It is a G-protein-coupled receptor that is expressed at high levels in BONE and in KIDNEY. Parathyroid Hormone Receptor Type 1,PTH-PTHrP Receptor,PTH-Related Peptide Receptor,PTH-Related Protein Receptor,PTHLP Receptor,PTHrP Receptor,Parathyroid Hormone Receptor,Parathyroid Hormone Receptor 1,Parathyroid Hormone Receptor Type I,Parathyroid Hormone-Like Peptide Receptor,Parathyroid Hormone-Related Peptide Receptor,Receptor, Parathyroid Hormone-Like Peptide,PTH PTHrP Receptor,PTH Related Peptide Receptor,PTH Related Protein Receptor,Parathyroid Hormone Like Peptide Receptor,Parathyroid Hormone Related Peptide Receptor,Receptor, PTH-PTHrP,Receptor, PTH-Related Peptide,Receptor, PTH-Related Protein,Receptor, PTHLP,Receptor, PTHrP,Receptor, Parathyroid Hormone
D019390 Arrestins Regulatory proteins that down-regulate phosphorylated G-protein membrane receptors, including rod and cone photoreceptors and adrenergic receptors.

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