Fluorescent Labeling of Peptides
ChemFiles Volume 5 Article 12
Labeling peptides with fluorescent dyes or other labels provides powerful tools for the investigation of biological relevant interactions like receptor-ligand-binding,1-3 protein structures,4-6 and enzyme activity.
Fluorescence energy transfer (FRET) between a donor and an acceptor label is widely applied for such investigations. It can be determined by a number of different methods, e.g., quenching and other intensity measurements, donor or acceptor depletion kinetics, and fluorescence lifetime or emission anisotropy measurements.3-13 A variety of enzyme substrates have been designed and used,14-22 partially based on quenching of emission through a second label, that is eliminated through the separation of label and quencher by cleavage of substrate.
Labeled peptides can be prepared by either modifying isolated peptides or by incorporating the label during solid-phase synthesis. Three strategies are used to label peptides with dyes:
- Labeling during synthesis of peptide. Dyes that are not damaged by unblocking procedures are incorporated onto the amino terminus of the peptide chain.
- Synthetic peptides can be covalently modified on specific residues and labels incorporated following synthesis.
- Synthetic peptides may be covalently labeled by amine- or thiolreactive protein labels.
Fluorophores can be conjugated to the N-terminus of a resin-bound peptide before other protecting groups are removed and the labeled peptide is released from the resin. Amine-reactive fluorophores are used in about 5-fold molar excess relative to the amines of the immobilized peptide. Reactive fluorescein, sulforhodamine B, tetramethylrhodamine, coumarin, eosin, dabcyl, dabsyl, or biotin labels, as well as several of our new atto labels, should be stable enough to resist the harsh deprotection conditions. Dabcyl has been frequently used as quencher. Another possibility is the use of fluorescence or chromophore labeled amino acids to incorporate labels at specific sites of peptides.
Labeling can also be achieved indirectly by using a biotinylated amino acid. If, for example, Fmoc-Lys(biotinyl)-OH, no. 73749 is used in peptide synthesis, the biotin group allows specific binding of streptavidin or avidin-conjugate to that site. A variety of fluorophores are available as (strept)avidin conjugates
Following the routine synthesis procedure, peptides can also be labeled by practically all labels used for protein labeling. This means mainly amine reactive labels, or thiol reactive labels, if a cystein has been used for the peptide. Whereas the common standard procedures for protein labeling are based on aqueous solutions of target proteins, labeling peptides in organic solvents like DMSO or DMF requires specific modifications. Use of triethylamine can be added to ensure that the target amino groups of the peptide are deprotonated, which is required for the labeling procedure.
- Kenworthy, A. K. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 2001, 24, 289.
- Hoppe, A.; Christensen, K.; Swanson, J. A. Imaging protein-protein interactions in living cells. Biophys. J. 2002, 83, 3652.
- Ozawa, T.; Umezawa, Y. Peptide assemblies in living cells. Methods for detecting protein-protein interactions. Supramol. Chem. 2002, 14, 271.
- Peled, H.; Shai, Y. Synthetic S-2 and H-5 segments of the Shaker K+ channel: secondary structure, membrane interaction, and assembly within phospholipid membranes. Biochemistry 1994, 33, 7211.
- Ben-Efraim, I.; Strahilevitz, J.; Bach, D.; Shai, Y. Secondary structure and membrane localization of synthetic segments and a truncated form of the IsK (minK) protein. Biochemistry 1994, 33, 6966.
- Marmé, N.; Knemeyer J. P.; Sauer, M.; Wolfrun, J. Inter- and Intramolecular Fluorescence Quenching of Organic Dyes Tryptophan. Bioconjugate Chem. 2003, 14, 1133.
- Wieb van der Meer, B.; Coker, G.; Simon, C. Resonance Energy Tranfer: Theory and Data. VCH: New York, 1994.
- Young, R. M.; Arnette, J. K.; Roess, D. A.; Barisas, B. G. Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics. Biophys. J. 1994, 67, 881.
- Chicester, U. K.; Andrews D. L.; Demidov A. A. Resonance Energy Transfer. Wiley & Sons: New York, 1999.
- Widengren, J.; Schweinberger, E.; Berger, S.; Seidel, C. A. M. Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy: theory and experimental realizations. J. Phys. Chem. A 2001, 105, 6851.
- Clayton, A. H. A.; Hanley, Q. X. Arndt-Jofin D. J.; Subramaniam, V.; Jovin, T. M. Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM) Biophys. J. 2002, 83, 1631.
- Clegg, R. M; Holub, O.; Gohlke, C. Fluorescence lifetime-resolved imaging: measuring lifetimes in an image. Methods Enzymol. 2003, 360, 509.
- Jares-Erijman, E. A.; Jovin, T. M. FRET imaging. Nat. Biotechnol. 2003, 21, 1387–1395.
- Matayoshi, E. D.; Wang, G. T.; Krafft G. A.; Erickson, J. Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. Science 1990, 247, 954.
- Wang, G. T. Design and Synthesis of New Fluorogenic HIV Protease Substrates Based on Resonance Energy Transfer. Tetrahedron Lett. 1990, 31, 6493.
- Garcia-Echeverria, C.; Rich, D. H. New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain. FEBS Lett. 1992, 297, 100.
- Wang, G. T.; Krafft, G. A. Automated Synthesis of Fluorogenic Protease Substrates: Design of Probes for Alzheimers Disease-Associated Proteases. Bioorg. Med. Chem. Lett. 1992, 2, 1665.
- Maggiora, L. L.; Smith, C. W.; Zhang, Z. Y. A general method for the preparation of internally quenched fluorogenic protease substrates using solid-phase peptide synthesis. J. Med. Chem. 1992, 35, 3727.
- Contillo, L. G., et al. General Strategy for the Synthesis of Eosin Fluorescein Energy Transfer Substrates for High Sensitivity Screening of Protease Inhibitors. In Techniques in Protein Chemistry V, Crabb J. W., Ed. Academic Press: New York, 1994; pp 493.
- Weder, J. K. P; Kaiser, K-P. Fluorogenic Substrates for Hydrolase Detection Following Electrophoresis. J. Chromatogr. A 1995, 698, 181.
- Cavrois, M.; de Noronah, C.; Greene, W. C. Nat. Biotechnol. 2002, 20, 1151–1154.
- Marmé, N.; Knemeyer, J. P.; Wolfrun, J.; Sauer, M.; Highly Sensitive Protease Assay Using Fluorescence quenching of Peptide Probes Based on Photoinduced Electron Transfer. Angew. Chem., Int. Ed. Engl. 2004, 43, 3798.