Open AccessFluorescent Labeling of Specific Cysteine Residues Using CyMPL
Author(s) -
Puljung Michael C.,
Zagotta William N.
Publication year - 2012
Publication title -
current protocols in protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.409
H-Index - 32
eISSN - 1934-3663
pISSN - 1934-3655
DOI - 10.1002/0471140864.ps1414s70
Subject(s) - cysteine , chemistry , covalent bond , residue (chemistry) , fluorescence , amino acid , electrophile , metal ions in aqueous solution , cysteine metabolism , combinatorial chemistry , stereochemistry , biochemistry , metal , organic chemistry , enzyme , physics , quantum mechanics , catalysis
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 ( C ysteine M etal P rotection and L abeling), which enables specific labeling by incorporating a cysteine of interest into a minimal binding site for group 12 metal ions (e.g., Cd 2+ and Zn 2+ ). 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.