Experimental characterization of the denatured state ensemble of proteins.
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The traditional view of the denatured state ensemble of proteins is that it behaves as a classic random coil. This model has important implications for the analysis of protein stability, protein folding, and cooperativity; namely that the effects of mutations on the free energy of the denatured state ensemble can be ignored. This assumption, which is still routinely made, at least at the implicit level, greatly simplifies the analysis of such experiments. However it has long been recognized that the denatured state ensemble (DSE) of real proteins is often quite different from a random coil and can exhibit significant structural preferences. In some cases parts of the chain can even adopt relatively well-defined conformations, particularly under native conditions. Well-studied examples of DSE interactions include elements of hydrogen-bonded secondary structure, particularly helices or turns, as well hydrophobic clusters, hydrophobic aromatic clusters, and more recently interactions involving charged residues. Deviations from random-coil behavior are of practical importance if they influence protein folding, stability, or function, or if they compromise our analysis and interpretation of experiments. The existence of residual structure in the DSE naturally leads to the question of its role in protein folding and stability, and raises the possibility that some mutations could exert a significant part of their effect by altering the DSE. Much of our understanding of the interactions governing protein stability and the folding process have been generated by mutational studies; thus, a detailed understanding of the denatured state ensemble is critical.