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 (T _g) 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 (PSD) 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 ΔC _p0(T _g) 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 T _g is systematically decreased. We find that the heat capacity increment from the TMDSC trace is linearly well correlated (negatively) with beta-sheet content φ _C determined from FSD, yielding ΔC _p = 0.475-0.494φ _C. The correlation allows the beta-sheet content to be determined from a direct measurement of the heat capacity increment at T _g. 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.