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To detect the local structural change in an interface between proteins induced by the substrate binding and dissociation, a solvatochromic fluorescent N-L-alanyl-5-(N,N-dimethylamino)-naphthalene-1-sulfonamide (DanAla) was introduced into 132 position of the dimer interface in BamHI. Before addition of the substrate, the fluorescence from the normal planer excited state of DanAla moiety was observed as a main emission, and thereby the DanAla in the dimer interface is located in the hydrophobic microenvironment. The incubation with the substrate for 20 min induced the gradual increase in fluorescence intensity around 430 nm. The fact reflects that the polarity is reduced by the slight structural change initiated by the formation of the complex with the substrate. Furthermore, the incubation for more than 20 min caused the slight decrease in fluorescence around 430 nm and an appearance of fluorescence (560 nm) due to twisted intramolecular charge transfer (TICT) excited state. Therefore, the DanAla is exposed to comparative polar environment after the dissociation of the substrate. The fluorescence lifetime as a minor component, which is attributed to the TICT excited state, was reduced by addition of the substrate. The results provide that the hydrophobicity in the dimer interface is increased by the substrate binding. Interestingly, we found that the structure of an initial form is different from that of a refolded form after the dissociation of the substrate using a spectral subtraction technique. We have achieved detection of the changing structure induced by the substrate binding and dissociation using a steady-state and time-resolved fluorescence. [ABSTRACT FROM AUTHOR]

A light-driven artificial molecularnanomachine was constructed based on DNA scaffolding. Pyrene-modifiedwalking strands and disulfide bond-connected stator strands, employedas anchorage sites to support walker movement, were assembled intoa 2D DNA tile. Pyrene molecules excited by photoirradiation at 350nm induced cleavage of disulfide bond-connected stator strands, enablingthe DNA walker to migrate from one cleaved stator to the next on theDNA tile. The time-dependent movement of the walker was observed andthe entire walking process of the walker was characterized by distributionof the walker-stator duplex at four anchorage sites on the tile under different irradiation times.Importantly, the light-fuelled mechanical movements on DNA tile werefirst visualized in real time during UV irradiation using high-speedatomic force microscopy (HS-AFM). [ABSTRACT FROM AUTHOR]