6 Cocoon Silk: From Mesoscopic Materials Design …
259
6.3.4 Level Four Structure of SF Materials: Fishnet-Like
Crystallite Networks and Nanofibrils
Merely crystalline binding is insufficient to construct tough materials. A tough material depends to a considerable extent on how these β-crystallites are connected. This
is then associated with the fourth level of structure: β-crystallite networks. In general,
the level four structures of a protein should refer to complexes composed of multiple
subunits. The molecular β-crystallite networks of SF materials can be defined as a
fourth-level structure [16, 18, 32]. Notably, each individual nanofibril is a crystallite
networks in which amorphous chains link the crystallites together. In this regard, we
can also regard the nanofibril (crystal network) as a level four structure. Nanofibrils
are observed in all forms of SF materials (including fibers, hydrogels, films, and
scaffolds), indicating that nanofibrils are the basic mesoscopic structural units of the
hierarchical structure of SF materials.
In terms of the type of crystal network topology, recently, atomic force microscopy
(AFM) has become a powerful tool for examining the nanostructures within nanofibrils by measuring the corresponding nanomechanical performance. Liu et al. [18]
investigated the structure of nanofibrils obtained either from RSF solution or
from natural SF fiber via AFM imaging, small angle X-ray scattering (SAXS),
Fourier transform infrared (FTIR) spectroscopy, and XRD characterization and
confirmed that these two types of nanofibrils share great similarities in structure
and morphology. This similarity enables the use of individual RSF nanofibrils as
an effective surrogate for natural SF fibers. After their initial investigation, they
further used the regenerated nanofibrils to verify the type of topology of the βcrystallite network using AFM force spectroscopy [18]. By studying the unfolding
force patterns of β-crystallites, Liu et al. presented a hypothesis about the mechanism
by which the breakage of β-crystallites occurs during stretching and concluded that
the β-crystallites are associated with each other in a nano-fishnet topology [18]. In the
AFM force spectroscopy experiments, the AFM tip was used to pull out individual
nanofibrils in order to probe the elasticity of β-crystallites. The manner in which
the silk protein chains are connected via β-crystallites can significantly affect the
dissipation of force from the AFM tip within the semi-crystalline networks; this is
reflected by the measured force patterns. Typical sawtooth patterns were observed in
the force versus extension trajectories of the regenerated nanofibrils. In these force
patterns, the height of the force peaks corresponds to the strength of the hydrogen
bonds between the β-strands in the β-sheet and/or the inter-β-sheet interactions in
the β-crystallites, while the changes in the level of extension between two adjacent
peaks correspond to the released length of the polypeptides (contour length changes
can be determined by fitting data to the worm-like chain model). The aforementioned
sawtooth patterns of SF nanofibrils are distinct from the characteristic plateau force
pattern observed for amyloid fibrils [35], indicating that the molecular network structures within amyloid fibrils and SF nanofibrils are different. This disparity between
the amyloid fibrils and SF nanofibrils was also observed while examining the XRD
259
6.3.4 Level Four Structure of SF Materials: Fishnet-Like
Crystallite Networks and Nanofibrils
Merely crystalline binding is insufficient to construct tough materials. A tough material depends to a considerable extent on how these β-crystallites are connected. This
is then associated with the fourth level of structure: β-crystallite networks. In general,
the level four structures of a protein should refer to complexes composed of multiple
subunits. The molecular β-crystallite networks of SF materials can be defined as a
fourth-level structure [16, 18, 32]. Notably, each individual nanofibril is a crystallite
networks in which amorphous chains link the crystallites together. In this regard, we
can also regard the nanofibril (crystal network) as a level four structure. Nanofibrils
are observed in all forms of SF materials (including fibers, hydrogels, films, and
scaffolds), indicating that nanofibrils are the basic mesoscopic structural units of the
hierarchical structure of SF materials.
In terms of the type of crystal network topology, recently, atomic force microscopy
(AFM) has become a powerful tool for examining the nanostructures within nanofibrils by measuring the corresponding nanomechanical performance. Liu et al. [18]
investigated the structure of nanofibrils obtained either from RSF solution or
from natural SF fiber via AFM imaging, small angle X-ray scattering (SAXS),
Fourier transform infrared (FTIR) spectroscopy, and XRD characterization and
confirmed that these two types of nanofibrils share great similarities in structure
and morphology. This similarity enables the use of individual RSF nanofibrils as
an effective surrogate for natural SF fibers. After their initial investigation, they
further used the regenerated nanofibrils to verify the type of topology of the βcrystallite network using AFM force spectroscopy [18]. By studying the unfolding
force patterns of β-crystallites, Liu et al. presented a hypothesis about the mechanism
by which the breakage of β-crystallites occurs during stretching and concluded that
the β-crystallites are associated with each other in a nano-fishnet topology [18]. In the
AFM force spectroscopy experiments, the AFM tip was used to pull out individual
nanofibrils in order to probe the elasticity of β-crystallites. The manner in which
the silk protein chains are connected via β-crystallites can significantly affect the
dissipation of force from the AFM tip within the semi-crystalline networks; this is
reflected by the measured force patterns. Typical sawtooth patterns were observed in
the force versus extension trajectories of the regenerated nanofibrils. In these force
patterns, the height of the force peaks corresponds to the strength of the hydrogen
bonds between the β-strands in the β-sheet and/or the inter-β-sheet interactions in
the β-crystallites, while the changes in the level of extension between two adjacent
peaks correspond to the released length of the polypeptides (contour length changes
can be determined by fitting data to the worm-like chain model). The aforementioned
sawtooth patterns of SF nanofibrils are distinct from the characteristic plateau force
pattern observed for amyloid fibrils [35], indicating that the molecular network structures within amyloid fibrils and SF nanofibrils are different. This disparity between
the amyloid fibrils and SF nanofibrils was also observed while examining the XRD