Progress in Materials Science 76 (2016) 229–318
`
`Contents lists available at ScienceDirect
`
`Progress in Materials Science
`
`j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p m a t s c i
`
`Keratin: Structure, mechanical properties,
`occurrence in biological organisms, and efforts
`at bioinspiration
`
`Bin Wang, Wen Yang, Joanna McKittrick, Marc André Meyers
`
`University of California, San Diego, La Jolla, CA 92093-0418, United States
`
`⇑
`
`a r t i c l e
`
`i n f o
`
`a b s t r a c t
`
`Article history:
`Received 9 February 2015
`Received in revised form 8 May 2015
`Accepted 13 May 2015
`Available online 4 July 2015
`
`Keywords:
`Keratins and keratinous materials
`Bioinspiration
`Biochemistry
`Filament-matrix structure
`Mechanical property
`
`A ubiquitous biological material, keratin represents a group of
`insoluble, usually high-sulfur content and filament-forming pro-
`teins, constituting the bulk of epidermal appendages such as hair,
`nails, claws, turtle scutes, horns, whale baleen, beaks, and feathers.
`These keratinous materials are formed by cells filled with keratin
`and are considered ‘dead tissues’. Nevertheless, they are among
`the toughest biological materials, serving as a wide variety of inter-
`esting functions, e.g. scales to armor body, horns to combat aggres-
`sors, hagfish slime as defense against predators, nails and claws to
`increase prehension, hair and fur to protect against the environ-
`ment. The vivid inspiring examples can offer useful solutions to
`design new structural and functional materials.
`Keratins can be classified as a- and b-types. Both show a charac-
`teristic filament-matrix structure: 7 nm diameter intermediate fil-
`aments for a-keratin, and 3 nm diameter filaments for b-keratin.
`Both are embedded in an amorphous keratin matrix. The molecular
`unit of intermediate filaments is a coiled-coil heterodimer and that
`of b-keratin filament is a pleated sheet. The mechanical response of
`a-keratin has been extensively studied and shows linear Hookean,
`yield and post-yield regions, and in some cases, a high reversible
`elastic deformation. Thus, they can be also be considered ‘biopoly-
`mers’. On the other hand, b-keratin has not been investigated as
`comprehensively. Keratinous materials are strain-rate sensitive,
`and the effect of hydration is significant.
`Keratinous materials exhibit a complex hierarchical structure:
`polypeptide chains and filament-matrix structures at the nanoscale,
`
`⇑ Corresponding author.
`
`E-mail address: mameyers@eng.ucsd.edu (M.A. Meyers).
`
`http://dx.doi.org/10.1016/j.pmatsci.2015.06.001
`0079-6425/Ó 2015 Elsevier Ltd. All rights reserved.
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`230
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`B. Wang et al. / Progress in Materials Science 76 (2016) 229–318
`
`organization of keratinized cells into lamellar, tubular–intertubular,
`fiber or layered structures at the microscale, and solid, compact
`sheaths over porous core, sandwich or threads at the macroscale.
`These produce a wide range of mechanical properties: the Young’s
`modulus ranges from 10 MPa in stratum corneum to about 2.5 GPa
`in feathers, and the tensile strength varies from 2 MPa in stratum
`corneum to 530 MPa in dry hagfish slime threads. Therefore, they
`are able to serve various functions including diffusion barrier,
`buffering external attack, energy-absorption,
`impact-resistance,
`piercing opponents, withstanding repeated stress and aerodynamic
`forces, and resisting buckling and penetration.
`A fascinating part of the new frontier of materials study is the
`development of bioinspired materials and designs. A comprehen-
`sive understanding of the biochemistry, structure and mechanical
`properties of keratins and keratinous materials is of great impor-
`tance for keratin-based bioinspired materials and designs. Current
`bioinspired efforts including the manufacturing of quill-inspired
`aluminum composites, animal horn-inspired SiC composites, and
`feather-inspired interlayered composites are presented and novel
`avenues for
`research are discussed. The first
`inroads into
`molecular-based biomimicry are being currently made, and it is
`hoped that this approach will yield novel biopolymers through
`recombinant DNA and self-assembly. We also identify areas of
`research where knowledge development is still needed to elucidate
`structures and deformation/failure mechanisms.
`Ó 2015 Elsevier Ltd. All rights reserved.
`
`Contents
`
`1.
`2.
`
`3.
`
`2.3.
`
`Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
`Structure, biochemistry and properties of a- and b-keratins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
`2.1.
`Classification of keratin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
`Basic structural characteristics of a- and b-keratins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
`2.2.
`2.2.1.
`Filament-matrix structure at nanoscale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
`2.2.2. Molecular structure and formation of the filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
`Biochemistry of a- and b-keratins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
`Biochemical and molecular analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
`2.3.1.
`Solubility and amino acid compositions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
`2.3.2.
`2.3.3.
`Biosynthesis of keratins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
`2.3.4.
`Formation of keratinous materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
`2.4. Mechanical properties of a- and b-keratins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
`Two-phase model for a-keratin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
`2.4.1.
`The a-helix to b-sheet transition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
`2.4.2.
`2.4.3.
`Viscoelasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
`2.4.4. Hydration sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
`Keratin research history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
`2.5.
`Structure and mechanical properties of keratinous materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
`Keratinous materials based on a-keratin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
`3.1.
`Stratum corneum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
`3.1.1.
`3.1.2. Wool and hair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
`3.1.3.
`Quills . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
`3.1.4. Horns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
`3.1.5. Hooves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
`3.1.6.
`Nails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
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`231
`
`3.3.
`
`3.2.
`
`3.1.7. Whale baleen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
`3.1.8. Hagfish slime threads. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
`3.1.9. Whelk egg capsules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
`Keratinous materials based on b-keratin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
`3.2.1.
`Feathers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
`3.2.2.
`Beaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
`3.2.3.
`Claws . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
`Keratinous materials based on a- and b-keratin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
`3.3.1.
`Reptilian epidermis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
`3.3.2. Hard and soft epidermis of testudines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
`3.3.3.
`Pangolin scales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
`Bioinspired designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
`4.1.
`Traditional bioinspiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
`4.2. Molecular-based bioinspiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
`Conclusions and critical assessment of field. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
`Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
`
`4.
`
`5.
`
`1. Introduction
`
`Nature presents a plethora of unique materials that have evolved for billions of years and have
`become a continuing source of inspiration for engineers. Biomimetics, the science of imitating nature,
`is thus an exciting field where the evolutionary refinements are investigated and the biological solu-
`tions are applied to develop new materials. The study of biological materials, Biological Materials
`Science, indispensably paves the way for inventing novel materials by providing principles and mech-
`anisms obtained from natural designs [1–8]. The more traditional approach is being complemented by
`molecular biomimetics, which shows a bright potential [9].
`Many biological materials are composites based on biopolymers and some minerals. This combina-
`tion yields materials with outstanding properties and functionalities, considering the mainly weak
`constituents (primarily C, O, N, H, Ca, P and S). Others process nanoscale fibrils with high tensile
`strength. Wood may have a strength per unit weight comparable to that of the strongest steels; spider
`silk has a higher specific strength and modulus than steels; shell, bone, and antler have a toughness an
`order of magnitude greater than their mineral constituents (e.g. calcite, hydroxyapatite) [10]. The
`secret for achieving this is usually the hierarchically organized structure incorporating biopolymers
`and minerals.
`Keratin represents the most abundant structural proteins in epithelial cells [11], and together with
`collagen, is the most important biopolymer in animals [12]. According to the Ashby map [13], shown
`in Fig. 1, keratin is among the toughest biological materials, possessing both high toughness and high
`modulus, although it is solely composed of polymeric constituents, and seldom contains minerals [14].
`Keratinous materials, formed by specifically organized keratinized cells filled with mainly fibrous pro-
`teins (keratins), are natural polymeric composites that exhibit a complex hierarchical structure rang-
`ing from nanoscale to centimeter scale: polypeptide chain structure, filament-matrix structure,
`lamellar structure, sandwich structure. They compose the hard integuments of animals, e.g. epidermis,
`wool, quills, horns of mammals, as well as feathers, claws and beaks of birds and reptiles, and effec-
`tively serve a variety of functions, such as for protection and defense, predation and as armor. There-
`fore, a thorough understanding of the relationships between the units that make up a functional
`keratinous material would expectantly provide useful knowledge in designing new materials.
`Keratinous materials have started to trigger great interest in recent years, and the nascent research
`area of bioinspiration is gaining increasing attention. However, there have only been very few reports
`on keratin in terms of biological and structural features, e.g. the classic books including Mercer [15]
`and Fraser et al. [16] on keratins and Feughelman [17] on a-keratin, and two review papers covering
`their structure, mechanical properties [12] and phase transition-induced elasticity of a-helical bioe-
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`Fig. 1. Materials property chart for biological materials: toughness versus Young’s modulus [13].
`
`lastomers [18]. Here, our aim is to provide a present-day comprehensive review of keratins and ker-
`atinous materials, incorporating biological and materials science perspectives to illustrate the struc-
`tural designs and functional properties in order to stimulate the development of novel bioinspired
`keratin-based designs.
`
`2. Structure, biochemistry and properties of a- and b-keratins
`
`Keratins refer to a group of insoluble and filament-forming proteins produced in certain epithelial
`cells of vertebrates; they belong to the superfamily of intermediate filament proteins [19], and form
`the bulk of the horny layer of the epidermis and the epidermal appendages such as hair, nails, horns,
`and feathers. These keratinous materials, having a high content of cysteine that distinguishes them
`from other proteins, are typically durable, tough and unreactive to the natural environment; they
`are assumed to provide mechanical support and diverse protective functions in the adaptation of ver-
`tebrates to the external environment [16,20].
`
`2.1. Classification of keratin
`
`Keratins and keratinous materials are often discussed in terms of a- and b-keratins [21]. Based on
`X-ray diffraction, keratins can be classified into a-pattern, b-pattern, feather-pattern and amorphous
`pattern [16,22–25]. The feather pattern has been considered as b-pattern since both show the same
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`233
`
`characteristic reflections, which has been well-accepted [26]. The amorphous pattern represents the
`component of the amorphous matrix (detailed in Section 2.2.1) in a-keratinous tissues [27]. Because
`the ordered structures (a- or b-patterns) dominate the X-ray diffraction, keratinous materials are con-
`veniently distinguished by these ordered components. Additionally, the two regular secondary struc-
`tures, a-helices and b-sheets, are the two major internal supportive structures in proteins [28]; thus,
`they are usually used to classify keratins.
`Fig. 2 shows the wide-angle X-ray diffraction patterns of these two types of keratins: the a-keratin
`gives a pattern with an equatorial reflection of spacing 0.98 nm (this corresponds to the distance
`between a-helical axes) and a meridional reflection of spacing 0.515 nm (relates to the a-helix pitch
`projection). The b-keratin has a prominent axial repeat of 0.31 nm reflection (the distance between
`residues along the chain in a b-sheet), the 0.47 nm equatorial arc (the distance between chains in
`a b-sheet) and the broad equatorial reflection at 0.97 nm (corresponds to intersheet distance)
`[16,24,29,30]. a-keratin is found in mammals (there is one mammal, the pangolin, that is reported
`to have both a and b), and it is the primary constituent of wool, hair, nails, hooves, horns and the stra-
`tum corneum (outermost layer of skin). The b-form is the major component of hard avian and reptilian
`tissues, such as feathers, claws and beaks of birds, and scales and claws of reptiles [31], listed in
`Table 1. Wool, as a representative a-keratin material, has been extensively studied, as well as feathers
`as a typical b-keratin material. Wool and feathers will be discussed as representatives of a-keratin and
`b-keratin, respectively, in Section 2.3.2.
`In addition, there are other classifications being used in the literature. In terms of modes of biosyn-
`thesis [32] and the amount of sulfur cross links [15], keratins can be classified as soft keratins (e.g.
`stratum corneum) usually weakly consolidated and with a lower amount of sulfur and lipids, and hard
`keratins found in hair, nails, claws, beaks, quills, which have a more coherent structure and a higher
`amount of sulfur [16]. Keratins are also discussed in terms of mammalian keratin, reptilian keratin and
`avian keratin. Besides, studies on keratinization in vertebrates and the evolution of epidermal proteins
`have considered keratins as true keratin (a-keratin) and corneous beta-proteins (b-keratins) [33,34].
`
`Fig. 2. X-ray diffraction patterns of (a) a-keratin and (b) b-keratin [16].
`
`Table 1
`Distribution of a- and b-keratin.
`
`a-Keratin
`
`b-Keratin
`a- and b-Keratin
`
`Wool, hair, quills, fingernails, horns, hooves; stratum corneum
`
`Feathers, avian beaks and claws, reptilian claws and scales
`Reptilian epidermis, pangolin scales
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`2.2. Basic structural characteristics of a- and b-keratins
`
`2.2.1. Filament-matrix structure at nanoscale
`Both a- and b-keratinous materials show a fine filament-matrix structure at the nanoscale. Here
`the ‘filament’, for a-keratins, denotes the ‘intermediate filament (IF)’ which represents the structural
`feature seen by transmission electron microscopy and shows an intermediate size (7–10 nm in diam-
`eter) between two other major classes of filamentous structures: microfilaments (actin, 7 nm) and
`microtubules (24 nm) [35]. For b-keratins, the ‘filament’ is called ‘beta-keratin filament’ and has a
`diameter of 3–4 nm [26,27]. Fig. 3 presents
`transmission electron micrographs of
`the
`filament-matrix structure for typical a-keratinous (IFs in hair, Fig. 3a) and b-keratinous materials
`(beta-keratin filaments in feather rachis, Fig. 3b). Table 2 compares the major structural characteristics
`of a- and b-keratins. The filaments are ordered components composed by tightly bonded polypeptide
`chains and are considered as crystalline portions [29]. The a-keratin IF and the beta-keratin filament
`show different sizes and generate distinct X-ray diffraction patterns (seen in Fig. 2 and Table 2). In
`addition, the a-keratin has specialized constituent proteins: several kinds of low-sulfur proteins com-
`pose the IFs [36] while the matrix consists of high-sulfur and high-glycine–tyrosine proteins [16]. For
`b-keratin, there are no different types of proteins [16]; the filament and matrix are incorporated into
`one single protein [26]. Finally, the molecular mass of a-keratin ranges from 40 to 68 kDa, which is
`much larger than that of b-keratin, 10–22 kDa [37].
`
`Fig. 3. Transmission electron micrographs of typical keratinous materials with clear filament-matrix structure: (a) cross section
`of a human hair (a-keratin), stained with osmium tetroxide, showing 7 nm diameter intermediate filaments embedded in a
`darker matrix; (b) cross section of a seagull feather rachis (b-keratin), stained with potassium permanganate, showing the
`3.5 nm diameter b-keratin filaments differentiated by the densely stained matrix [16].
`
`Table 2
`Basic structures of a- and b-keratins.
`
`Similarity: structural feature
`
`Diameters of the filaments
`(nm)
`X-ray diffraction patterns
`[16,29]
`
`Constituting proteins
`
`Characteristic structure
`Molecular mass [37]
`
`a-Keratin
`
`b-Keratin
`
`Filament-matrix structure: IFs and beta-keratin filaments embedded in an amorphous
`matrix
`IFs and beta-keratin filaments generate characteristic X-ray diffraction patterns
`IFs: 7
`Beta-keratin filaments: 3–4
`
`Equatorial reflection with spacing 0.98 nm
`and a meridional reflection with spacing
`0.515 nm
`The IFs consist of several kinds of low-sulfur
`proteins [36], while the matrix consists of
`high-sulfur and high-glycine–tyrosine pro-
`teins [16]
`Based on a-helical structure
`40–68 kDa
`
`Axial repeat of 0.31 nm reflection and the
`equatorial reflection 0.47 nm
`
`Do not have two different types of
`proteins [16]; the filament and matrix are
`incorporated into one single protein [26]
`
`Based on b-pleated sheet structure
`10–22 kDa
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`
`2.2.2. Molecular structure and formation of the filaments
`The differences of molecular structure and formation of the filaments are the most important fea-
`tures that distinguish a- and b-keratins [28,38–40], shown in Figs. 4 and 5. The a-keratin proteins are
`organized as coiled coils. The a-helix conformation for the polypeptide chains was first postulated
`independently by Pauling and Crick [41,42], shortly after Pauling, Corey, and Branson [43] identified
`the structure as consisting of two helically wound chains of polypeptides. Naturally occurring
`
`Fig. 4. Intermediate filament structure of a-keratin: (a) ball-and-stick model of the polypeptide chain, and a-helix showing the
`location of the hydrogen bonds (red ellipse) and the 0.51 nm pitch of the helix [44]; (b) schematic drawing of the intermediate
`filament formation (reproduced based on [28,45]): a-helix chains twist to form the dimers, which assemble to form the
`protofilament. Four protofilaments organize into the intermediate filament.
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`B. Wang et al. / Progress in Materials Science 76 (2016) 229–318
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`Fig. 5. Structure of the beta-keratin filaments: (a) ball-and-stick model of the polypeptide chain, and illustration of the pleated
`beta-sheet [44]); (b) schematic drawing of the formation of beta-keratin filament (adapted from [16]): one polypeptide chain
`folds to form four b-strands which twist to form the distorted b-sheet. Two sheets assemble to form a beta-keratin filament.
`
`a-helices found in proteins are all right-handed. The helical structure is stabilized by the hydrogen
`bonds (red circled line in Fig. 4a, [44]) inside the helix chain, causing the chain to twist and exhibit
`a helical shape. Fig. 4b shows the IF formation process [28,45]: two isolated right-handed a-helix
`chains form a left-handed coiled-coil, the dimer (45 nm long), by disulfide cross links; then dimers
`aggregate end-to-end and stagger side-by-side via disulfide bonds [46] to form a protofilament (about
`2 nm diameter); two protofilaments laterally associate into a protofibril; four protofibrils combine
`into a circular or helical IF with a diameter of 7 nm. It is clear that the IF is based on coiled-coil struc-
`ture. Then, the IFs pack into a supercoiled conformation, and link with the matrix proteins. The
`sulfur-rich amorphous keratin matrix consists of protein chains that have a high amount of cysteine
`residues or high amounts of glycine, tyrosine and phenylalanine residues [47].
`The molecular structure and assembly mechanisms of IF proteins, which a-keratins belong to, can
`be found in the literature [19]. Although there has not been a high-resolution characterization of ker-
`atin IFs, recent studies have reported the crystal structure within the heterodimeric coiled-coil region
`[48]. Keratins are expected to share structural homology with vimentin, an IF protein, and the crystal
`structure of vimentin in the literature [49,50] can provide useful information to the understanding of
`keratin structure. In addition to keratin, fibrin and myosin also form IFs.
`For b-keratin, the pleated-sheet (Fig. 5a, [44]) consists of laterally packed b-strands which can be
`parallel or antiparallel (more stable), and the chains are held together by intermolecular hydrogen
`bonds (red circled line in Fig. 5a). The pleated sheet structure is stabilized by two factors: the hydro-
`gen bonds between beta strands contribute to forming a sheet and the planarity of the peptide bond
`forces a b-sheet to be pleated [28]. The formation of beta-keratin filament involves (Fig. 5b): the cen-
`tral region of one polypeptide chain folds to form four lateral beta-strands which then link through
`hydrogen bonding, resulting in a pleated sheet; then, the sheet distorts to lie in a left-handed helical
`ruled surface; each residue (marked by red circle in Fig. 5b) is represented by a sphere in the model
`(red dot in Fig. 5b); two pleated sheets are related by a horizontal diad, superpose and run in opposite
`directions, forming the filament with a diameter of 4 nm (a pitch length of 9.5 nm and four turns per
`unit). The terminal parts (not shown in Fig. 5b) of the peptide chains wind around the b-keratin fila-
`ments and form the matrix [26]. Therefore, keratins can be considered as a polymer/polymer compos-
`ite of crystalline filaments embedded in an amorphous matrix.
`
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`237
`
`2.3. Biochemistry of a- and b-keratins
`
`2.3.1. Biochemical and molecular analysis
`The systematic protein biochemical analyses of human cells and tissues revealed the diversity of
`human keratin polypeptides [51–53]; these proteins were separated into type I (acidic) and type II
`(basic to neutral) keratins. A new consensus nomenclature for mammalian keratin genes and proteins
`to accommodate functional genes and pseudogenes was developed, and it classifies the 54 functional
`keratin genes as epithelial and hair keratins (28 type I keratin genes with 17 epithelial and 11 hair ker-
`atins, and 26 type II keratin genes with 20 epithelial and 6 hair keratins) [20].
`a-keratin can only constitute its filamentous state through the coiled coil assembly and,
`heteropolymeric pair formation of type I and type II (1:1) protein molecules [19,54]. This gives the
`name, heterodimer (same as the dimer in Fig. 4b), which is the monomeric unit of the keratin IF
`(shown in Fig. 6a, [11,38]); it consists of two chains. Each one contains a central alpha-helical rod
`(about 46 nm in length) with non-helical C- and N-terminal regions [55,56]. The central rod region
`contains non-helical links at L1, L12, L2 and a stutter. The C- and N-terminal domains are involved
`in bonding with other IF molecules and matrix.
`For b-keratin, the unit molecule of the filaments also consists of three domains: the central domain
`with residues forming b-sheet and the N- and C-terminal domains (seen in Fig. 6b, the lower sche-
`matic) with different lengths and compositions depending on specific keratinous tissues [16,26,57].
`The central domain has been the focus in the literature for the molecular structure of b-keratin fila-
`ment. It is the central part of one polypeptide chain folding several times that forms a pleated sheet
`structure, the region within two dotted lines shown in Fig. 6b. The other two parts of the chain form
`the N- and C-terminal domains [26].
`
`Fig. 6. Detailed structure of: (a) molecular unit of a intermediate filament: the heterodimer. The non-helical N- and C-terminal
`domains bond with other intermediate filaments and matrix; the central region (about 46 nm in length) has the a-helical coiled
`coil segments (1A, 1B, 2A, 2B). There are short links (L1, L12 and L2) and a ‘stutter’ in middle segment (adapted from [11,38]);
`(b) molecular unit of b-keratin filament: the upper illustrates the distorted sheet and the lower is a schematic representation of
`a molecule with central domain and N- and C-terminal domains. The central domain (about 34 residues in length) consists of b-
`forming residues; the N- and C-terminal domains vary among species (adapted from [16,26]).
`
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`B. Wang et al. / Progress in Materials Science 76 (2016) 229–318
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`The molecular units, dimensions and keratin assemblies are summarized in Table 3. For a- and
`b-keratins, the unit molecules, heterodimer and one distorted pleated sheet, respectively, contain a
`central domain and two terminal domains. The central helical region of the a-keratin molecular unit
`contains about 33–35 residues and the non-helical N- and C-terminal domains contain about 136 resi-
`dues [58]. The length of the central region is about 45 nm [59] and the diameter about 2 nm [35]. For
`the b-keratin, the length of central region is about 2.3 nm and the diameter about 2 nm [16]. The ker-
`atin assembly for a-keratin involves the organization of dimers into IFs, the terminal domains link
`with other molecules and matrix proteins, and the terminal domains and matrix proteins wind around
`IFs to form keratin [26,60]. While for b-keratin, the pleated sheets arrange into filaments, C- and
`N-terminal domains compose the matrix and wind central domain, forming the keratin [26].
`
`2.3.2. Solubility and amino acid compositions
`Keratins are naturally insoluble due to intermolecular disulfide linkages [16], intramolecular disul-
`fide linkages [61], and interchain peptide linkages [62,63]. Table 4 lists the purification procedures
`developed to obtain keratin derivatives. For a-keratinous materials, reduction, oxidation and sulfitol-
`ysis methods have been used to generate satisfactory amounts of the derivatives [16,64–67]; while for
`b-keratinous materials, which have not been as extensively investigated as a-keratin, alkaline thiogly-
`collate and a combination of a disulfide bond-breaking reagent and a protein denaturant were
`described in literature [68,69]. There are also reports discussing degraded keratins produced by partial
`hydrolysis (with acid, alkali or enzymes) of wool, hair and feathers. The keratin fragments from
`hydrolysis are used in the manufacture of cosmetics, artificial leather and filaments [70]. For amino
`acid analysis, the acid hydrolysis of proteins and automated ion-exchange chromatography are used
`routinely [71]. The residue percent of wool (representing a-keratin) and feathers (representing
`b-keratin) are summarized in Table 5 [16,72,73]. It is clear that both show high content of half cystine
`(cysteine plus half cystine), which provides the disulfide bonds and distinguishes keratin as
`high-sulfur protein from other biopolymers. Whole wool shows a higher residue percent of half
`cystine and glutamic acid than whole feather rachis. The higher contents of glycine, proline and serine
`
`Table 3
`Comparison of a- and b-keratin: molecular unit (MU), dimension and keratin assembly.
`
`Molecular unit
`Residue number of MU
`
`Length of central MU
`Diameter of MU
`Keratin assembly
`
`a-Keratin
`
`b-Keratin
`
`Dimer
`Helical: 33–35
`Non-helical: about 136 [58]
`About 45 nm [59]
`Around 2 nm [35]
`Dimers organize into IFs; C-, N-terminal
`domains link with other molecules and
`matrix proteins, and these wind around
`IFs to form keratin [16,60]
`
`Distorted pleated sheet
`Pleated sheet forming: 34
`Non-sheet forming: 59–168 [26,57]
`2.3 nm [16]
`2 nm [16]
`Pleated sheets arrange into IFs; C- and N-
`terminal domains compose matrix, link
`with other molecules and wind central
`domain to form keratin [26]
`
`Table 4
`Solubility of a- and b-keratin.
`
`a-Keratin
` Reduction: by potassium thioglycollate in urea to obtain
`80–97% keratin from horn, hoof, hair, and further by
`starch-gel electrophoresis into high-sulfur and low-
`sulfur fractions [16,64,65]
` Oxidation: By treating wool with peracetic acid and
`dilute alkali [66]
`Sulfitolysis: By sodium bisulfite with urea and an
`oxidizing agent [67]
`
`b-Keratin
`