Fluorescence Evidences for Non-Homogeneity and Residual Structure of Denatured States

Abstract

About 30 % of human proteins do not fold into a stable 3D arrangement of secondary structure elements, but stay predominantly unfolded -similar to proteins under highly denaturing conditions. These proteins are involved in many cell signaling processes. Their characterization poses a great challenge for current experimental methods as they consist of an ensemble of rapidly interconverting conformations. Intense debate exists on the possibility that they show, to certain extent, residual structure, which might facilitate folding or enhance ligand binding. To study the unfolded state conformational heterogeneity using Forster resonance energy transfer (FRET), we used the lysozyme from the phage T4 (T4L) in denaturing conditions as a model system. We built an elastic network model that spans T4L's topology in order to evaluate local and global conformational changes by combining ensemble (ensemble time-resolved fluorescence lifetime and anisotropy) and single-molecule spectroscopic (multiparameter fluorescence detection, photon distribution analysis, (filtered) fluorescence correlation spectroscopy) methods. Through extensive comparison of models, we identified regions with apparent residual structure under highly denaturing conditions, which might serve as folding nuclei; and additionally we showed that chemically denatured T4L is not a random coil as previously thought. By using obtained distance restraints we determined that denatured T4L shows a native-like mean structure, albeit larger in size compared to the native state. We demonstrate here the necessity of careful data interpretation, but also the potential of a multidimensional approach to characterize an ensemble of states, which can be applied generally to unstructured or denatured proteins.