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W. Qiu and X.-Y. Liu
6.4.1.3 Raman Spectroscopy
Other than FTIR, Raman spectroscopy is another powerful and nondestructive technique used to investigate the secondary structures of SF materials [49, 50]. In general,
Raman spectroscopy can provide information that is complementary to that of FTIR
spectroscopy. As discussed, the signals in the FTIR spectrum correspond to the
absorbance energy of the infrared photons released by molecules. In contrast, the
Raman effect deals with the scattering process involving interactions between the
incident photons and the sample molecules; in other words, the Raman spectra record
the inelastically scattered energy of photons. For molecules to be infrared-active,
molecular vibrations must initiate a change in the dipole moment of the molecules.
To determine the Raman activity, the molecular vibrations should induce a change
in the molecular polarizability. In principle, if a molecule has a center of symmetry,
then a Raman-active vibration is always infrared-inactive, and vice versa [54]. For
example, polarizable bonds such as C–C, S–S, N=N, and O–O bonds can display
intense Raman bands; however, in the infrared spectrum, these bonds show only
weak or even undetectable bands. Another advantage of Raman spectroscopy for
studying protein molecular structure is that it can also provide quantitative information about the content of secondary structures in a manner similar to the FTIR
method (Fig. 6.18b).
Recently, a Raman spectromicroscopy technique was developed by mounting a
microscope onto the conventional Raman spectroscopy setup. This technique can
precisely refine the size of an incident laser beam to less than 20 μm and can thus
collect scattered signals from very small samples, making it an ideal tool for recording
the high-quality Raman spectra of single silk filaments. Moreover, by combining
Raman spectromicroscopy with an external mechanical deformation puller, the transition process of secondary structures in silk filaments when subjected to mechanical
stretching can be detected in situ. Specifically, it has been reported that silkworm silks
display well-defined Raman spectra in which the frequencies of some bands shift
under the action of tensile stress or strain, suggesting a molecular conformation transition. For instance, Raman microspectroscopy has been used to quantitatively study
the effect of mechanical deformation on the secondary structure conformation and
order parameters of Samia cynthia ricini (S. c. ricini) silk fibroin fibers. According
to a study by Rousseau et al. [55] samples were obtained from an aqueous solution
stored in the silk gland and stretched at draw ratios (λ) ranging from 0 to 11. The
Raman data unambiguously show that in response to mechanical deformation, SF
molecules undergo a cooperative α-helix to β-sheet conformational transition.
Based on the FTIR and Raman data, it is surprising that the amounts of β-sheets
in silkworm silk fibers (~50%) nearly coincide with the proportion of relevant amino
acid sequences (i.e., GAGAGS amino acids that are recognized to be involved in
β-sheet generation), which is 53%. In comparison, for Nephila dragline silk fibers,
the amino acids (A) n constitute only 18% of the total content, and this is significantly
lower than the β-sheet content (36–37%) measured by Raman spectroscopy. In the
AG and GGA motifs, which are usually located adjacent to the (A) n blocks, the sum
value increases to 31%. Thus, this result strongly suggests that apart from the poly
W. Qiu and X.-Y. Liu
6.4.1.3 Raman Spectroscopy
Other than FTIR, Raman spectroscopy is another powerful and nondestructive technique used to investigate the secondary structures of SF materials [49, 50]. In general,
Raman spectroscopy can provide information that is complementary to that of FTIR
spectroscopy. As discussed, the signals in the FTIR spectrum correspond to the
absorbance energy of the infrared photons released by molecules. In contrast, the
Raman effect deals with the scattering process involving interactions between the
incident photons and the sample molecules; in other words, the Raman spectra record
the inelastically scattered energy of photons. For molecules to be infrared-active,
molecular vibrations must initiate a change in the dipole moment of the molecules.
To determine the Raman activity, the molecular vibrations should induce a change
in the molecular polarizability. In principle, if a molecule has a center of symmetry,
then a Raman-active vibration is always infrared-inactive, and vice versa [54]. For
example, polarizable bonds such as C–C, S–S, N=N, and O–O bonds can display
intense Raman bands; however, in the infrared spectrum, these bonds show only
weak or even undetectable bands. Another advantage of Raman spectroscopy for
studying protein molecular structure is that it can also provide quantitative information about the content of secondary structures in a manner similar to the FTIR
method (Fig. 6.18b).
Recently, a Raman spectromicroscopy technique was developed by mounting a
microscope onto the conventional Raman spectroscopy setup. This technique can
precisely refine the size of an incident laser beam to less than 20 μm and can thus
collect scattered signals from very small samples, making it an ideal tool for recording
the high-quality Raman spectra of single silk filaments. Moreover, by combining
Raman spectromicroscopy with an external mechanical deformation puller, the transition process of secondary structures in silk filaments when subjected to mechanical
stretching can be detected in situ. Specifically, it has been reported that silkworm silks
display well-defined Raman spectra in which the frequencies of some bands shift
under the action of tensile stress or strain, suggesting a molecular conformation transition. For instance, Raman microspectroscopy has been used to quantitatively study
the effect of mechanical deformation on the secondary structure conformation and
order parameters of Samia cynthia ricini (S. c. ricini) silk fibroin fibers. According
to a study by Rousseau et al. [55] samples were obtained from an aqueous solution
stored in the silk gland and stretched at draw ratios (λ) ranging from 0 to 11. The
Raman data unambiguously show that in response to mechanical deformation, SF
molecules undergo a cooperative α-helix to β-sheet conformational transition.
Based on the FTIR and Raman data, it is surprising that the amounts of β-sheets
in silkworm silk fibers (~50%) nearly coincide with the proportion of relevant amino
acid sequences (i.e., GAGAGS amino acids that are recognized to be involved in
β-sheet generation), which is 53%. In comparison, for Nephila dragline silk fibers,
the amino acids (A) n constitute only 18% of the total content, and this is significantly
lower than the β-sheet content (36–37%) measured by Raman spectroscopy. In the
AG and GGA motifs, which are usually located adjacent to the (A) n blocks, the sum
value increases to 31%. Thus, this result strongly suggests that apart from the poly