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

Abstract

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 (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 ∆C 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 ∆C 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.

Synthesis and Properties of Silk Fibroin/Konjac Glucomannan Blend Beads

“…If the CH 2 OH and COOH do not participate in the hydrogen bonding, the domain of the random coil conformation should be increased. However, as results show through the increasing of β-sheet, such groups may participate in the intermolecular bonds with the NH group of SF [33][34][35]. …”

Non-invasive characterization of structure and morphology of silk fibroin biomaterials using non-linear microscopy

“…cellular proliferation [1,5], differentiation [6][7][8], deposition of new structural proteins [9,10] and ultimately the regeneration of functional tissue [4,11,12]. Traditionally, methods such as NMR, FTIR and X-Ray spectroscopy have been used to assess biomaterials at the molecular level [13][14][15][16][17], while SEM and TEM have been invaluable tools for characterizing the three dimensional morphology of biomaterial scaffolds [5,[18][19][20][21][22][23]. Histology and immunostaining as well as assays for determining the expression levels of specific proteins are often used to assess how scaffolds interact with cells as tissues develop [5,10,18,20,[22][23][24].…”

“…The Bombyx mori silkworm silk protein, fibroin, can be described by two structural models: Silk I, consisting of type II β turn, random coil domains, and mixed structures including alpha helices, and Silk II, consisting mostly of antiparallel β pleated sheets. [14,15]. The β sheet content and the alignment of these β sheet crystals, along with the non-crystalline domains of the protein, are important determinants of the bulk mechanical properties and degradation kinetics of biomaterials generated from silk [14,36,37,[39][40][41].…”

Biocompatible silk fibroin scaffold prepared by reactive inkjet printing

“…FTIR spectra were taken of the amide I region (1705-1595 cm -1 ), which is most commonly used to determine the secondary structure of proteins as this region has less convolution of peaks than other areas of the spectra [2,20]. Films untreated with methanol had a peak at 1638 cm -1 , associated with random coils; however, with the increasing volumes of methanol, this peak shifts to 1620 cm -1 which is indicative of b-sheets [21], and in (Fig. 2a), this peak is marked with a vertical dashed line.…”

“…Fourier self deconvolution (FSD) of the amide I spectra was performed by OriginPro 2016 software using the technique previously described by Hu et al [21]. The newly found bands were used to identify different component secondary structures of each of the spectra.…”