Resolving the Heterogeneity of the Ensemble of Unfolded States by a Combination of Fluorescence Spectroscopic Methods

Abstract

Intense debate exists on the possibility that unfolded proteins show, to certain extent, residual secondary structure. Transient structure formation might facilitate folding (nucleation site) and/ or enhance binding to ligands. Some proteins such as those categorized as intrinsically disordered proteins take advantage of these features. Here, we used the model enzyme lysozyme from the phage T4 (T4L), a simple two subdomain protein, which already exists in an equilibrium of at least three conformations under native conditions. We created a set of 24 double mutants of the cysteine-free pseudo-wild type by inserting an unnatural amino acid and a cysteine mutation and site-specifically labeled them using orthogonal chemistry with a Forster resonance energy transfer (FRET) dye pair. Our set of variants allows us to build up a network of distances spanning the enzyme in order to monitor the ensemble of conformations. The behavior of the protein under highly denaturing conditions was observed by a combination of ensemble (ensemble time-resolved fluorescence lifetime and anisotropy) and single-molecule spectroscopic (multiparameter fluorescence detection, photon distribution analysis, (filtered) fluorescence correlation spectroscopy) methods. Our network covered all possible directions over the whole protein allowing us to map specifically the local motions (dye mobility) and global changes. We identified regions with residual structure which exist even under highly denaturing conditions. Additionally, the combination of ensemble and single-molecule methods allows us to resolve the full heterogeneity of the proteins’ denatured conformations.