Broadband Multidimensional Spectroscopy Identifies the Amide II Vibrations in Silkworm Films

Abstract

We used two-dimensional infrared spectroscopy to disentangle the broad infrared band in the amide II vibrational regions of Bombyx mori native silk films, identifying the single amide II modes and correlating them to specific secondary structure. Amide I and amide II modes have a strong vibrational coupling, which manifests as cross-peaks in 2D infrared spectra with frequencies determined by both the amide I and amide II frequencies of the same secondary structure. By cross referencing with well-known amide I assignments, we determined that the amide II (N-H) absorbs at around 1552 and at 1530 cm–1 for helical and β-sheet structures, respectively. We also observed a peak at 1517 cm−1 that could not be easily assigned to an amide II mode, and instead we tentatively assigned it to a Tyrosine sidechain. These results stand in contrast with previous findings from linear infrared spectroscopy, highlighting the ability of multidimensional spectroscopy for untangling convoluted spectra, and suggesting the need for caution when assigning silk amide II spectra.

Flow-Induced Protein Chain Deformation, Segmental Orientation, and Phase Separation in Native Silk Feedstock

“…The strongest component of the amide I band is oriented roughly perpendicular to the peptide chain backbone, while the strongest components of the amide II and III bands are roughly parallel to the chain. 113,119 Hence, these differences in relative intensities provided clear indications that at least some of the fibroin had been oriented with its chain axis in the flow direction, in agreement with the previous reports by Boulet-Audet et al 111,112 Further sudden changes occurred within a few seconds after the end of the flow period, with the amide peaks decreasing and the O−H stretching band increasing slightly. This produced sudden decreases in the amide I and II peaks in the difference spectra (Figure 9b,c), with corresponding increases in the water O−H stretching band.…”

“…Concurrently, a new peak appeared at 1695 cm –1 , while the amide II peak (the asymmetric combination of C–N stretching and N–H bending vibrations) appeared to move from 1545 to 1520 cm –1 (Figure e). These changes are consistent with the loss of random coil or helical structures in NSF and the fibroin developing an antiparallel β-sheet structure in response to flow. − ,− …”

“…In addition to the changes in peak height associated with microphase separation, changes in the apparent positions of the amide I and II difference peaks were observable (Figure e). In principle, peptide bands can be deconvoluted to provide structural information. − ,− In addition to chain conformation, however, it is expected that the peak positions in NSF are also affected by the presence of water, due to its dielectric constant and the strength of hydrogen bonding. − Moreover, the peak centered around 1640 cm –1 in the direct spectrum of NSF (Figure b,c) is expected to include at least two components due to the amide I and water bending bands. − It may be noted that additional absorbance due to water accounts for the intensity of the 1640 cm –1 peak being roughly twice that of the 1545 cm –1 peak; otherwise the amide I and II peaks of B. mori fibroin should be of similar height. ,, …”

Extracted Silk Fibroin‐Cellulose Acetate Nanofibrous Scaffolds for Tissue Engineering Applications

“…The peak in the region 1509–1511 cm −1 corresponded to the amide N─H of the β‐sheet crystalline structure of SF. [ 8 ] The ratio for amide I:amide II was 1.00:1.40 for native silk, 1.00:1.03 for degummed silk, and 1.00:0.20 for regenerated SF. The blue shift in amide I from degummed silk to regenerated SF showed the predominant random coil (amorphous) conformation.…”

“…This peak is associated with the amide II conformation of SF and therefore becomes more prominent when the sericin layer is eliminated. [ 8 ]…”