Solution structure of a protein denatured state and folding intermediate

Abstract

The most controversial area in protein folding concerns its earliest stages. Questions such as whether there are genuine folding intermediates, and whether the events at the earliest stages are just rearrangements of the denatured state or progress from populated transition states, remain unresolved. The problem is that there is a lack of experimental high-resolution structural information about early folding intermediates and denatured states under conditions that favour folding because competent states spontaneously fold rapidly. Here we have solved directly the solution structure of a true denatured state by nuclear magnetic resonance under conditions that would normally favour folding, and directly studied its equilibrium and kinetic behaviour. We engineered a mutant of Drosophila melanogaster Engrailed homeodomain that folds and unfolds reversibly just by changing ionic strength. At high ionic strength, the mutant L16A is an ultra-fast folding native protein, just like the wild-type protein; however, at physiological ionic strength it is denatured. The denatured state is a well-ordered folding intermediate, poised to fold by docking helices and breaking some non-native interactions. It unfolds relatively progressively with increasingly denaturing conditions, and so superficially resembles a denatured state with properties that vary with conditions. Such ill-defined unfolding is a common feature of early folding intermediate states and accounts for why there are so many controversies about intermediates versus compact denatured states in protein folding.

Identification and characterization of protein folding intermediates

“…Solving the structure of the intermediate/denatured state using standard NMR methods revealed that the conformation of helices 2 and 3 was highly native like, whereas helix 1 had secondary structure but no tertiary interactions (Figure 4) [53].…”

“…Overall, it appears that protein folding intermediates are stabilized both by native and non-native interactions [10,46,53].…”

“…As described below, this approach has been successfully employed to determine the structure of the folding intermediate of a small alpha-helical protein, the Engrailed homeodomain (En-HD) (Figure 4) [53].…”

Protein folding: Then and now

“…Under denaturing conditions, the protein can still exhibit native conformational bias and retain a certain amount of residual native structures. Mounting experimental evidence [168,[195][196][197][198] supports residual native-like structural elements in the denatured state for a variety of proteins. Using residual dipolar coupling from NMR measurements, Shortle and Ackerman [195] showed that native-like topology persists under strong denaturing conditions as high as 8 M Urea for a truncated staphylococcal nuclease.…”

“…In recent years, it has been found that even for the conventionally observed small two-state proteins (~100 amino acids or less) there exist partially unfolded intermediates on the folding pathways. These intermediates are generally undetectable in kinetic folding experiments [8,116,[164][165][166][167][168] and, therefore, are called "hidden intermediates". In addition, mounting evidence has indicated that the intermediate states formed during protein folding and unfolding may have significant roles in protein functions (Fig.…”