title: Fluorescent labeling of specific cysteine residues using CyMPL.
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.
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, Washington.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Puljung
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, Washington.
authors.forename: William N
authors.initials: WN
authors.lastname: Zagotta
mesh_terms: D000818:Animals; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D008958:Models, Molecular; D009865:Oocytes; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*; D029867:Xenopus Proteins / Q000737:chemistry; D014982:Xenopus laevis; D015032:Zinc / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D029867:Xenopus Proteins; D002104:Cadmium; D015032:Zinc; D003545:Cysteine