| Biophysical studies of proteins have traditionally focused solely on their folded, "active" conformations. The less well characterized unfolded state, however, is an equal partner in defining the free-energy of folding. What is more, as the starting point for the protein folding reaction, the structure of the unfolded state is a principle determinant of the folding mechanism. Consequently, a better understanding of the structure of the unfolded state may be critical to our understanding of both folding thermodynamics and kinetics. Unfortunately, the conformational heterogeneity of the unfolded state renders its study by traditional, biophysical methods extremely difficult or impossible. Here we present an overview of our current understanding of the structure of the unfolded state in addition to three novel studies into the properties and engineering applications of unfolded proteins. These studies investigate the extent to which putative residual denatured state structure affects the dimensional scaling of chemically unfolded proteins, the degree to which the dimensions of physiologically unfolded proteins are defined by sequence rather than bulk composition, and finally, a strategy is demonstrated for exploiting the sharp, all-or-nothing transition characteristic of the folding of small, single-domain proteins for the development of a novel class of ligand-induced folding-based protein biosensors. |
