Conformation transition kinetics of regenerated Bombyx mori silk fibroin membrane monitored by time-resolved FTIR spectroscopy

The ethanol-induced conformation transition of regenerated Bombyx mori silk fibroin membrane from a poorly defined to the well ordered state was monit ored by time-resolved Fourier transform infrared spectroscopy (FTIR) for th e first time. From the analysis of FTIR difference spectra, taken on time s cales as short as 6 s and up to 1 h after addition of ethanol. intensity vs . time plots of an increasing band at 1618 cm(-1) were observed indicating formation of a beta -sheet coincident with the loss of intensity of a band at 1668 cm(-1) indicating decreases of random coil and/or silk I structure. Both infrared markers were fitted with identical biphasic exponential deca y functions, however, there was a clear burst phase occurring prior to the onset of the observed transitions. The conformation transition process is i ndicated to either proceed sequentially through (at least) two intermediate states that contain different levels of beta -sheet structure or to have p arallel pathways of initial beta -sheet formation followed by a slower 'per fection' phase. The first observed process forms in a burst phase a few sec onds after mixing (or even faster), prior to the collection of the first sp ectrum at 6 s. The second observed process occurs with a time constant of s imilar to 0.5 min, the intermediate present at this stage then continues wi th a time constant of 5.5 min completing the observed formation of the beta -sheet. The conformation transition of this slower intermediate is not onl y indicated by an analysis of the kinetics of the random coil and beta -she et-specific bands discussed above. it roughly coincides with the appearance of an additional infrared marker at 1695 cm(-1), which may be a marker for p-sheet structure specific to the formation of the perfected structure. Th e conformation transition of this protein analyzed by infrared spectroscopy provides insight into a part of the fascinating process of cocoon formatio n in B. mori. (C) 2001 Elsevier Science B.V. All rights reserved.