Fluorescent Labeling of Specific Cysteine Residues Using CyMPL

Michael C. Puljung1, William N. Zagotta1

1 Department of Physiology and Biophysics, University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.14
DOI:  10.1002/0471140864.ps1414s70
Online Posting Date:  November, 2012
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Abstract

The unique reactivity and relative rarity of cysteine among amino acids makes it a convenient target for the site‐specific chemical modification of proteins. Commercially available fluorophores and modifiers react with cysteine through a variety of electrophilic functional groups. However, it can be difficult to achieve specific labeling of a particular cysteine residue in a protein containing multiple cysteines, in a mixture of proteins, or in a protein's native environment. This unit describes a procedure termed CyMPL (Cysteine Metal Protection and Labeling), which enables specific labeling by incorporating a cysteine of interest into a minimal binding site for group 12 metal ions (e.g., Cd2+ and Zn2+). These sites can be inserted into any region of known secondary structure in virtually any protein and cause minimal structural perturbation. Bound metal ions protect the cysteine from reaction while background cysteines are covalently blocked with non‐fluorescent modifiers. The metal ions are subsequently removed and the deprotected cysteine is labeled specifically. Curr. Protoc. Protein Sci. 70:14.14.1‐14.14.10. © 2012 by John Wiley & Sons, Inc.

Keywords: fluorescence; FRET; metal binding; cysteine modification

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Fluorescent Labeling of a Specific Cysteine in a Protein Containing Multiple Cysteines or in a Mixture of Soluble Proteins
  • Basic Protocol 2: Specific Labeling of an Extracellular Protein Domain in Xenopus Oocytes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fluorescent Labeling of a Specific Cysteine in a Protein Containing Multiple Cysteines or in a Mixture of Soluble Proteins

  Materials
  • Protein mixture with engineered metal‐binding site for the target cysteine (∼1 µM of the labeling target)
  • Labeling buffer (see recipe)
  • 1.1 mM CdCl 2 stock solution in H 2O
  • 10 mM N‐ethylmaleimide (NEM) in labeling buffer
  • 10 mM fluorescein‐5‐maleimide in labeling buffer
  • 500 mM ethylenediaminetetraacetic acid (EDTA) in water, pH 8.0
  • 1 mM glutathione (optional)

Basic Protocol 2: Specific Labeling of an Extracellular Protein Domain in Xenopus Oocytes

  Materials
  • OR2 solution (see recipe)
  • 10 mg/ml bovine serum albumin (BSA) in water
  • 10 mM CdCl 2 in water
  • Xenopus laevis oocytes (defolliculated and injected with RNA encoding the protein intended for labeling or sham‐injected) (Gordon and Zagotta, )
  • 10 mM NEM in OR2 solution
  • 500 mM EDTA in water, pH 8.0
  • 1 mM Alexa Fluor 546 in OR2 solution
  • 35‐mm polystyrene culture dishes
  • Glass Pasteur pipet, broken and fire‐polished
  • Orbital shaker
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Figures

Videos

Literature Cited

Literature Cited
   Gordon, S.E. and Zagotta, W.N. 1995. Localization of regions affecting an allosteric transition in cyclic nucleotide‐activated channels. Neuron 14:857‐864.
   Hermanson, G. 1996. Bioconjugate Techniques. Academic Press, San Diego, Calif.
   Islas, L.D. and Zagotta, W.N. 2006. Short‐range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence. J. Gen. Physiol. 128:337‐346.
   Kosower, N.S., Kosower, E.M., Newton, G.L., and Ranney, H.M. 1979. Bimane fluorescent labels: Labeling of normal human red cells under physiological conditions. Proc. Natl. Acad. Sci. U.S.A. 76:3382‐3386.
   Kuiper, J.M., Pluta, R., Huibers, W.H., Fusetti, F., Geertsma, E.R., and Poolman, B. 2009. A method for site‐specific labeling of multiple protein thiols. Protein Sci. 18:1033‐1041.
   Lakowicz, J.R. 2006. Principles of Fluorescence Spectroscopy. Springer, New York.
   Puljung, M.C. and Zagotta, W.N. 2011. Labeling of specific cysteines in proteins using reversible metal protection. Biophys. J. 100:2513‐2521.
   Ratner, V., Kahana, E., Eichler, M., and Haas, E. 2002. A general strategy for site‐specific double labeling of globular proteins for kinetic FRET studies. Bioconjug. Chem. 13:1163‐1170.
   Rulisek, L. and Vondrasek, J. 1998. Coordination geometries of selected transition metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Hg2+) in metalloproteins. J. Inorg. Biochem. 71:115‐127.
   Smith, J.J., Conrad, D.W., Cuneo, M.J., and Hellinga, H.W. 2005. Orthogonal site‐specific protein modification by engineering reversible thiol protection mechanisms. Protein Sci. 14:64‐73.
   Taraska, J.W. and Zagotta, W.N. 2010. Fluorescence applications in molecular neurobiology. Neuron 66:170‐189.
   Taraska, J.W., Puljung, M.C., Olivier, N.B., Flynn, G.E., and Zagotta, W.N. 2009a. Mapping the structure and conformational movements of proteins with transition metal ion FRET. Nat. Methods 6:532‐537.
   Taraska, J.W., Puljung, M.C., and Zagotta, W.N. 2009b. Short‐distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions. Proc. Natl. Acad. Sci. U.S.A. 106:16227‐16232.
   Zheng, J. and Zagotta, W.N. 2000. Gating rearrangements in cyclic nucleotide‐gated channels revealed by patch‐clamp fluorometry. Neuron 28:369‐374.
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