In Situ Identification of Secondary Structures in Unpurified Bombyx mori Silk Fibrils Using Polarized Two-Dimensional Infrared Spectroscopy

Abstract

The mechanical properties of biomaterials are dictated by the interactions and conformations of their building blocks, typically proteins. Although the macroscopic behavior of biomaterials is widely studied, our understanding of the underlying molecular properties is generally limited. Among the noninvasive and label-free methods to investigate molecular structures, infrared spectroscopy is one of the most commonly used tools because the absorption bands of amide groups strongly depend on protein secondary structure. However, spectral congestion usually complicates the analysis of the amide spectrum. Here, we apply polarized two-dimensional (2D) infrared spectroscopy (IR) to directly identify the protein secondary structures in native silk films cast from Bombyx mori silk feedstock. Without any additional peak fitting, we find that the initial effect of hydration is an increase of the random coil content at the expense of the helical content, while the β-sheet content is unchanged and only increases at a later stage. This paper demonstrates that 2D-IR can be a valuable tool for characterizing biomaterials.

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

“…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. ,, …”

Structural adaptability and surface activity of tardigrade-inspired peptides

“…This problem can be partially overcome by 2D-IR spectroscopy. (30) The combination of CD and (2D-)IR spectroscopy is thus a powerful tool to characterize the structure of the peptides.…”

“…To overcome these problems, we use two-dimensional infrared spectroscopy (2D-IR). Compared to the linear infrared spectra, 2D-IR better resolves the individual vibrational bands because the 2DIR signal scales quadratically with the absorption cross-section (while the conventional IR signal scales linearly with the cross-section), leading to narrower peaks and a removal of the solvent background(30, 32) In pump-probe 2D-IR spectroscopy, an intense, narrow-band infrared pump pulse (with adjustable center frequency ν pump ) resonantly excites at a specific frequency (in the present case, in the amide I band). A delayed, broad band probe pulse probes the frequency-dependent IR-absorption change Δ A , which results from the excitation by the pump beam.…”

Linearly Polarized Infrared Spectroscopy for the Analysis of Biological Materials

“…Nevertheless, a methodology based on the measurement of absorbance at multiple polarization angles was introduced for the analysis of polymers, 25 and has been shown to be applicable for biological samples. 6,26,27 Additionally, numerous instrumental and methodological advances have resulted in a plethora of available p-IR techniques, including polarized ATR Fourier transform infrared spectroscopy (p-ATR FT-IR), 28 p-IR microscopy, 6,29,30 polarization-dependent atomic force microscopy–infrared spectroscopy (AFMIR), 31 optical photothermal IR (O-PTIR), 32 polarized two-dimensional IR, 33 p-IR dual comb polarimetry, 34 and IR ellipsometry, 35 many of these are discussed later in this review.…”