Abstract：A combinational study of circular dichroism，intrinsic fluorescence of protein and exogenous fluorescence probe of ANS was made to investigate the conformational change of silk fibroin in methanol-water mixtures as well as the mechanism.The spectral results showed that small hydrophobic regions were formed in silk fibroin in methanol-water mixtures at the concentration lower than 30% (V/V) via hydrophobic interaction，which was decreased at higher methanol content due to a structural transition of silk fibroin from random coil to β-sheet.The conformational change of silk fibroin was found to be of a close relationship with the microstructure of the solvent and to be determined by the interaction between the peptide unit of silk fibroin and the cluster of the mixed solvent.Methanol-water mixture at low concentration had little effect on the solvation of the peptide unit and the conformation of silk fibroin，as a consequence of the fact that the inherent water structure was conserved.The transition from the tetrahedral-like water structure to the chain-like methanol structure，due to the increasing concentration of methanol，induced the conformational change of silk fibroin to eliminate the contact of peptide unit with the solvent molecular.
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