Fluorescent Labeling of Specific Cysteine Residues Using CyMPL

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 CdCl₂ stock solution in H₂ O
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 CdCl₂ 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

Figure 14.14.1 Schematic diagram depicting the specific cysteine protection method, CyMPL. The group 12 metal ion (M²⁺ ) binding affinity of a desired cysteine (uncircled C) is increased by placing an additional metal‐binding amino acid nearby [shown here as histidine (H)]. M²⁺ is used to protect the desired cysteine (i ) while the background cysteines (in circles) are reacted with a non‐fluorescent modifying reagent such as N ‐ethylmaleimide (NEM) (ii ). Upon M²⁺ removal with a chelator (e.g., EDTA; iii ), the specific cysteine is available to react with the fluorophore (F; iv ). Adapted from Puljung and Zagotta ().

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Figure 14.14.2 Cd²⁺ affinity of helical peptides with introduced minimal metal binding sites. Concentration‐response curve showing the rate (normalized to 0 Cd²⁺ ) of the reaction of Cys‐only peptide and Cys/Cys i+3 peptide with bimane C₃ ‐maleimide as a function of [CdCl₂ ]_total . The dashed box marks a concentration range within which single cysteines are not expected to be bound to Cd²⁺ , but a site consisting of two cysteines should be fully bound. Affinity was assessed by examining the concentration‐dependent reduction of the rate of reaction of the peptides with bimane C₃ ‐maleimide. n = 5 to 6 for each concentration. Adapted from Puljung and Zagotta ().

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Figure 14.14.3 Metal‐binding sites. The cartoons depict the three possible cysteine‐histidine binding sites for group 12 metal ions. Cys/His i+3 and Cys/His i+4 are helical peptides with histidines placed three and four positions away from a cysteine, respectively. HCN S563C,K565H depicts one β‐strand from the C‐terminal fragment of the mouse HCN2 ion channel with a cysteine and histidine placed two residues apart.

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Figure 14.14.4 Dibromobimane reacted with two cysteines. Cartoon depicts the bifunctional fluorophore dibromobimane reacted with two cysteines spaced three residues apart on a model α‐helix.

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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.
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Fluorescent Labeling of Specific Cysteine Residues Using CyMPL

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited