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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 b-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.

The thermodynamic effects of the disulfide bond of the fragment protein of the starch-binding domain of Aspergillus niger glucoamylase was investigated by measuring the thermal unfolding of the wild-type protein and its two mutant forms, Cys3Gly/Cys98Gly and Cys3Ser/Cys98Ser. The circular dichroism spectra and the thermodynamic parameters of binding with beta-cyclodextrin at 25 degrees C suggested that the native structures of the three proteins are essentially the same. Differential scanning calorimetry of the thermal unfolding of the proteins showed that the unfolding temperature t1/2 of the two mutant proteins decreased by about 10 degrees C as compared to the wild-type protein at pH 7.0. At t1/2 of the wild-type protein (52.7 degrees C), the mutant proteins destabilized by about 10 kJ mol(-1) in terms of the Gibbs energy change. It was found that the mutant proteins were quite stabilized in terms of enthalpy, but that a higher entropy change overwhelmed the enthalpic effect, resulting in destabilization.

&tractNew surfactant ruthenium complexes, [Ru(X)(Y)(L8 or 12)Jn+, were synthesized by the reaction of [R~CI~(q~-p-cymene)]~ with L8 or 12 in methanol or DMSO, where L8 or 12 stands for tetradentate 6,6'-bis(N-octyl-or lau~lbenzimidazolyl)-2,2'-bipyridine. The complexes formed stable monolayer films on the Langmuir trough at the air-water interface and were transferred to a glass plate. The K-A isotherms indicate the phase transition between expanded liquid state and the solid state at around 14 mNm-', and monolayer collapses at a pressure of 45 mNm-'. This phase transition may be interpreted in terms of a change of molecular orientation. The structure of the LB films on glass were discussed.

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