Infrared spectroscopy of CT in the amide I region. The top panel (a) presents the second derivative of the absorbance spectrum (black) and the respective fit (red) for the dried drop of CT. The bottom panel (b) presents the absorbance spectra and the fitted component bands. The IR spectra corroborates the secondary structure change upon transition from soluble CT domain to solid CT silk fiber.

METHODS

Attenuated total reflection (ATR) FTIR spectroscopy was conducted on the CT fibers using a Vertex 70 FTIR Spectrometer (Bruker) equipped with an HgCdTe detector and continuously purged with dry air to minimize water vapor interference. The samples were positioned on the diamond crystal of a Bruker Platinum ATR unit and pressed onto the crystal with a piston in case of the fiber sample. The total spectral interval analyzed was between 3900 and 900 cm-1, the spectral resolution was 4 cm-1, and the zero-filling factor 2.
For the analysis, the amide I region (1700–1600 cm-1) was selected, residual water vapor signals (when necessary) and a baseline were subtracted (straight lines with baseline points at 1800, 1740, 1720, and 1580 cm−1). IR spectra were collected and pre-processed using the OPUS software (Bruker). Further evaluation was performed with the program Kinetics, kindly provided by E. Goormaghtigh (Université Libre de Bruxelles, Belgium). 
For secondary structure analysis, component bands were simultaneously fitted to the absorbance spectrum and its secondary derivative in the 1700-1590 cm−1 range. The second derivative spectra were calculated using a Savitzky-Golay window of 21 points and were weighted with a factor of 300 in the fit. Initially, five bands with mixed Gaussian/Lorentzian band shapes were fitted in the 1710-1590 cm−1 range. However, the optimal fit was obtained using three bands in the low wavenumber -sheet region, i.e. in total seven bands in the 1710-1590 cm−1 range (no baseline was fitted). The secondary structure content was estimated from the relative band areas assigned to the different secondary structures. The data were plotted using the software OriginPro.

The spectral ranges used for the assignment of secondary structures were: β-sheets 1610–1641 cm–1, anti-parallel β-sheets 1700-1690 cm–1, α-helixes 1648–1657 cm–1, and other structures 1642–1686 cm–1 (i.e. all other secondary structures: irregular, turns, bends, other helix types). Secondary structures other than α-helixes or β-sheets are expected to produce broad bands, which are relatively weak in second derivative spectra. Therefore the 1650 cm–1 band was assigned to α-helixes. 



FILE FORMAT
.csv text data

