Manipulating and Labeling of Proteins by Selectively Addressable Thiol Moieties

Henning D. Mootz

Henning D. Mootz

University of Münster, Institute of Biochemistry, Corrensstr. 36, 48149 Münster, Henning.Mootz@uni-muenster.de

Introducing fluorophores, biophysical probes and other defined chemical modifications into proteins is an enabling technology for their biophysical, biochemical and cellular investigations. Cysteine is arguably the most versatile proteinogenic amino acid in terms of both the importance for protein structure and catalysis and the chemical reactivity of the thiol side chain. Classical thiol bioconjugation with reagents like maleimides provides unique opportunities for the chemo- and regioselective introduction of various labels into proteins. However, its scope is severely restricted for proteins containing an essential or multiple cysteines. Bioorthogonal reactions can circumvent these limitations, but they require the incorporation of non-natural reactive groups, which often amounts to elaborate and expensive procedures and requires specialized chemical synthesis skills. Therefore, we have sought to expand the scope of the established thiol bioconjugation chemistry with its plethora of commercially available reagents at reasonable costs. Split inteins reconstitute proteins from two segments by protein trans-splicing. We have explored short sequence tags with a pre-modified single cysteine that can be appended by protein trans-splicing to a purified protein or to a protein in a living cell. Furthermore, we have developed non-natural amino acids with caged thiol side chains that can be selectively deprotected and hence allow for a defined labeling of two thiol groups in one protein, for example. Homocysteine with its altered reactivity of the thiol moiety further serves to manipulate and probe structural and catalytic properties of proteins.

 

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