Kinetically trapped metastable intermediate of a disulfide-deficient mutant of the starch-binding domain of glucoamylase.

Refolding of a thermally unfolded disulfide-deficient mutant of the starch-binding domain of glucoamylase was investigated using differential scanning calorimetry, isothermal titration calorimetry, CD, and (1)H NMR. When the protein solution was rapidly cooled from a higher temperature, a kinetic intermediate was formed during refolding. The intermediate was unexpectedly stable compared with typical folding intermediates that have short half-lives. It was shown that this intermediate contained substantial secondary structure and tertiary packing and had the same binding ability with beta-cyclodextrin as the native state, suggesting that the intermediate is highly-ordered and native-like on the whole. These characteristics differ from those of partially folded intermediates such as molten globule states. Far-UV CD spectra showed that the secondary structure was once disrupted during the transition from the intermediate to the native state. These results suggest that the intermediate could be an off-pathway type, possibly a misfolded state, that has to undergo unfolding on its way to the native state.