Population and structure determination of hidden folding intermediates by native-state hydrogen exchange-directed protein engineering and nuclear magnetic resonance.

Structural characterization of folding intermediates has been one of the important steps toward understanding the mechanism of protein folding. However, it has been very difficult to obtain high-resolution structures of folding intermediates. Such results have become available only very recently. Here, we review a procedure that uses the native-state amide hydrogen exchange-directed protein engineering method to populate partially unfolded intermediates and multidimensional NMR to solve the high-resolution structures of the intermediates.