Although the structure of native and native-like states can be determined using x-ray crystallography and in solution using NMR, little detailed information exists on the actual structure of denatured and intermediate states. Intermediate states are difficult to trap in a way that allow details structural analysis. In contrast to the "native" state which consists of an ensemble of closely related states, intermediates and denatured states would consist of an ensemble of many different states, making structural analysis more difficult. Religa and others from Fersht's lab have engineered a mutant of the engrailed homeodomain (En-HD) from Drosophila melanogaster that allows such structural analyses to be performed. The mutation, Leu16Ala (L16A), destabilizes the protein such that it can be denatured simply by changing ionic strength. It is stable at high ionic strength and folds quickly under those condition. However at physiological ionic strength it is "denatured" but contains significant alpha-helical structure but has nonnative contacts. It behaves like an early folding intermediate in that if placed in solutions of higher ionic strength it rearranges to form the native state. If placed in lower ionic strength, it progressively "unfolds" to yet other states. Given the ambiguities in how to define denatured and early folding intermediates states, Ferscht's group suggest an "explicit" definition of the denatured state. They define the unfolded state (U) as the "maximally unfolded state of a protein, in which the backbone NH groups have little protection against 1H/2H exchange". They define the denatured state, D, as the "lowest energy non-native state under a defined set of conditions". In this scenario, the denatured state could also be a folding intermediate if placed in conditions that promote folding. Previous work from the group showed that the denatured state of En-HD has three helices protected from 2H exchange and was one kcal/mol lower in energy than the unfolded state.
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