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Updated: Feb 6, 2026

Identifying Protein-protein Interaction Sites Using Peptide Arrays
Published on: November 18, 2014
Rational design of a highly reactive dicysteine peptide tag for fluorogenic protein labelling
Miroslava Strmiskova1, Kelvin Tsao, Jeffrey W Keillor
1Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie-Curie, Ottawa, ON K1N 6N5, Canada. jkeillor@uottawa.ca.
Abstract:
Rationally designed libraries of a short helical peptide sequence containing two cysteine residues were screened kinetically for their reactivity towards complementary dimaleimide fluorogens. This screening revealed variant sequences whose reactivity has been increased by an order of magnitude relative to the original sequence. The most reactive engineered sequences feature mutant residues bearing positive charges, suggesting the pKa values of the adjacent thiol groups have been significantly lowered, through electrostatic stabilization of the thiolate ionization state. pH-Rate profiles measured for several mutant sequences support this mechanism of rate enhancement. The practical utility of the enhanced reactivity of the final engineered dicysteine tag ('dC10*') was then demonstrated in the fluorogenic intracellular labelling of histone H2B in living HeLa cells.
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