Rational Design of a Highly Reactive Dicysteine Peptide Tag For Fluorogenic Protein Labelling [@theKeillors]

Miroslava Strmiskova,  Kelvin Tsao  and  Jeffrey W Keillor
Org. Biomol. Chem., 2018
doi: 10.1039/C8OB01417E

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 a specific protein in living cells.