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Issue 34, 2018
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Rational design of a highly reactive dicysteine peptide tag for fluorogenic protein labelling

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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.

Graphical abstract: Rational design of a highly reactive dicysteine peptide tag for fluorogenic protein labelling

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Publication details

The article was received on 14 Jun 2018, accepted on 14 Aug 2018 and first published on 14 Aug 2018


Article type: Paper
DOI: 10.1039/C8OB01417E
Citation: Org. Biomol. Chem., 2018,16, 6332-6340

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    Rational design of a highly reactive dicysteine peptide tag for fluorogenic protein labelling

    M. Strmiskova, K. Tsao and J. W. Keillor, Org. Biomol. Chem., 2018, 16, 6332
    DOI: 10.1039/C8OB01417E

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