Determining Beta-Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy

We report a study of self-assembled beta-pleated sheets in B. mori silk fibroin films using thermal analysis and infrared spectroscopy. B. mori silk fibroin may stand as an exemplar of fibrous proteins containing crystalline beta-sheets. Materials were prepared from concentrated solutions (2−5 wt % fibroin in water) and then dried to achieve a less ordered state without beta-sheets. Crystallization of beta-pleated sheets was effected either by heating the films above the glass transition temperature (Tg) and holding isothermally or by exposure to methanol. The fractions of secondary structural components including random coils, alpha-helices, beta-pleated sheets, turns, and side chains were evaluated using Fourier self-deconvolution (FSD) of the infrared absorbance spectra. The silk fibroin films were studied thermally using temperature-modulated differential scanning calorimetry (TMDSC) to obtain the reversing heat capacity. The increment of the reversing heat capacity ΔCp0(Tg) at the glass transition for the less ordered, noncrystalline, silk fibroin is found to be 0.478 ± 0.005 J/(g K). As crystalline beta-sheets form, the heat capacity increment at Tg is systematically decreased. We find that the heat capacity increment from the TMDSC trace is linearly well correlated (negatively) with beta-sheet content φC determined from FSD, yielding ΔCp = 0.475−0.494φC. The correlation allows the beta-sheet content to be determined from a direct measurement of the heat capacity increment at Tg. This type of analysis can serve as an alternative to X-ray methods and may have wide applicability to other crystalline beta-sheet forming proteins.