Fluorescence-quenching probes can be used to study the dynamics, folding, and aggregation of appropriately labeled protein systems.  Valuable structural information can be extracted from distance-dependent quenching experiments; however, the structural resolution is often limited by the large size of the chromophore labels typically required for this technique.  One solution is to use smaller quenchers that do not significantly perturb native protein structure.  We have shown that backbone thioamides, which are single-atom substitutions of a peptide bond, quench a variety of fluorophores in a distance-dependent fashion through F�rster resonance energy transfer (FRET) or photoinduced electron transfer (PET) mechanisms.  Since thioamide analogs of the natural amino acids can be incorporated at almost any position in a protein sequence, they can be paired with bulkier fluorophores that perhaps can only be tolerated in a few positions in a protein.  We have used this method to monitor the binding of thioamide-containing peptides to calmodulin, the unfolding of the villin headpiece subdomain, aggregation of a-synuclein, and protease activity, among other applications.

Thioamides as fluorescence-quenching probes

245th National Meeting (2013)

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