ABSTRACT
2. Self-Assembly of Proteins into Various Nanoscale Architecture
Proteins can spontaneously assemble into various forms of higher-order architectures on the nanometer length scale under appropriate biochemical and physiological conditions. In cells, many structural proteins self-assemble into fi lament, network or elastic mesh, which constitutes mechanical supports of cells or guides cellular activity including signal transduction
and material transportation. Many of these structures were able to readily form in vitro given appropriate buffer conditions, making them suitable to various biomaterial applications (Galland et al. 2013, Hudalla et al.
2014, Reymann et al. 2010). The higher-order nanoarchitectures, such as helical assembly, icosahedral particle and elastic fi ber, often exhibit unique properties that may not be readily achieved by other materials. They can be extremely resilient and versatile. Supramolecular assemblies of proteins are, therefore, highly suited for rational re-engineering and could be used as three-dimensional supports of smart biomaterials with desired properties.
2.1 Helical Assembly
The architecture of eukaryotic cells is supported mechanically by a hierarchical nanostructure called cytoskeleton, which is self-assembled from a collection of structural proteins (Fletcher and Mullins 2010). The cytoplasm of eukaryotic cells is in constant motion as cellular organelles are transported continuously from place to place along the tracks of the cytoskeleton. The cytoskeleton is the higher-order protein assemblies that form the rails of the cell’s transportation system; many motor proteins move on the tracks of rails made from the different components of cytoskeleton. The cytoskeleton is composed mainly of three types of higher-order protein assemblies:
microtubules, microfi lament (actin) and intermediate fi laments. Each type of protein assembly functions as a polymerized complex composed of many identical subunit proteins.
Microtubules provide mechanical supports for cells. They are the strongest component of the cytoskeletal polymers (Weisenberg 1972).
Microtubules can resist strong compression. Microtubules are functionally required by cell migration, mitosis, gene regulation and morphogenesis.
Microtubules also have a major structural role in eukaryotic cilia and fl agella.
Cells rely on the dynamic assembly and disassembly of microtubules to reorganize the cytoskeleton quickly in response to environmental stimuli. This allows cells to take advantage of both the adaptability and strength of microtubules. Different cells can have unique organizations of microtubules to suit specifi c needs. Microtubules are hollow rigid tubes whose outer diameter is about 24 nm while the inner diameter is about 12 nm (Fig. 1a). The basic building block of microtubules is a protein called tubulin heterodimers. Tubulin heterodimer is made up of two protein subunits, α- and β-tubulin, each of which has a molecular weight of approximately 50 kDa. The α/β-tubulin dimers polymerize end-to-end into linear protofi laments that associate laterally to form a single microtubule, which can then be extended by the addition of more α/β-tubulin dimers. Typically, microtubules are formed by a parallel association of thirteen protofi laments, although microtubules composed of fewer or more protofi laments have been
observed in vitro. Microtubules have a distinct polarity that is critical for their biological function. Tubulin polymerizes end to end, with the β-subunits of one tubulin dimer contacting the α-subunits of the next dimer. Therefore, in a protofi lament, one end has only the α-subunits exposed while the other end has only the β-subunits exposed. These ends are designated the minus and plus ends, respectively. Although microtubule elongation can take place at both the plus and minus ends, it is more rapid at the plus end. The lateral association of the protofi laments creates a pseudo-helical structure. One turn of the helix contains 13 tubulin dimers, each from a different protofi lament.
Two distinct types of interactions take place between the subunits of lateral
Figure 1. Helical assembly of protein nanomaterials. (a) 3D cryo-electron microscopic reconstruction of microtubule decorated by Ndc80 kinetochore complex at 8.6 Å resolution (EMDataBank entry: EMD-5223) from two orthogonal perspectives (Alushin et al. 2010).
(b) 3D cryo-electron microscopic reconstruction of F-actin decorated with topomyosin at 3.7 Å resolution (EMDataBank entry: EMD-6124) (von der Ecken et al. 2015). (c) 3D cryo-electron microscopic reconstruction of dynactin at 3.5 Å resolution, which interacts with dynein and structurally resembles F-actin (EMDataBank entry: EMD-2857) (Urnavicius et al. 2015).
protofi laments within the microtubule, which are called the A-type and B-type lattices. In the A-type lattice, the lateral associations of protofi laments occur between adjacent α and β-tubulin subunits. Hence, an α-tubulin subunit from one protofi lament can interact with a β-tubulin subunit from an adjacent protofi lament. In the B-type lattice, the α and β-tubulin subunits from one protofi lament interact with the α and β-tubulin subunits from an adjacent protofi lament, respectively. The B-type lattice was suggested to be the primary arrangement within microtubules (Nogales 2000).
The second major component of the cytoskeleton is a globular multi- functional protein called actin that assembles into heli cal microfi lament (Gunning et al. 2015). The microfi lament measures approximately 7 nm in diameter with a helical pitch of 37 nm (Fig. 1b and 1c). The actin is found in all eukaryotic cells at concentrations of over 100 M. The mass of an actin protein is about 42-kDa. It is the monomeric subunit of two types of fi laments in cells: microfi laments, one of the three major components of the cytoskeleton, and thin fi laments, part of the contractile apparatus in muscle cells. The actin proteins are observed as either a free monomer called G-actin or as part of a linear polymer microfi lament called F-actin, both of which are essential for such important cellular functions as the mobility and contraction of cells during cell division. Actin participates in many important cellular processes, including muscle contraction, cell motility, mitosis, cytokinesis, vesicle and organelle movement, cell signaling, and the establishment and maintenance of cell junctions and cell shape. Many of these processes are mediated by extensive and intimate interactions of actin with cellular membranes. The actin fi laments also contribute to the mechanical support of cells, whose dynamic properties are mediated via assembly and disassembly of actin fi laments. The helical structure of actin fi lament are structurally polarized and tightly controlled within cells by a multitude of actin-binding proteins, such as profi lin, Arp2/3 complex, myosin, fl aming, villin, fi mbrin, and cofi lin. The actin-binding proteins regulate polymerization of new actin fi laments, prevent polymerization of actin monomers, control fi lament length, and crosslink actin fi laments.
The interaction of signal transduction pathways with actin-binding proteins provides a mechanism for controlling the dynamics and structure of cytoskeleton. In vertebrates, three main groups of actin isoforms, α, β, and γ have been identifi ed. The α-actins, found in muscle tissues, are a major constituent of the contractile apparatus. The β and γ-actins coexist in most cell types as components of the cytoskeleton, and as mediators of internal cell motility. The diverse range of structures formed by actin enables it to fulfi ll a large range of functions that are regulated through the binding of tropomyosin along the fi laments (Fig. 1b). The actin fi lament has been used as a versatile scaffold to develop novel materials, such as three-dimensional electrical connections (Galland et al. 2013, Reymann et al. 2010).
Intermediate fi laments are the third type of major components of the cytoskeleton and essential for maintenance of correct tissue structure and function (Fuchs and Cleveland 1998). They form robust networks of 10-nm- thick fi laments, which in diameter is intermediate between 7-nm actin fi laments and 25-nm microtubules. Most types of intermediate fi lament are in cytoplasm, but one type, called lamin, is in nucleus. The minimal building block of intermediate fi lament is a parallel dimer. The monomers are encoded by a large gene family and share a similar structure consisting of a central long α-helical domain divided into sections and fl anked by a head and a tail domain, which are subject to posttranslational modifi cations and regulate assembly by their phosphorylation. The dimers form antiparallel, tetrameric assembly units, which in vitro rapidly associate laterally and longitudinally to form a 10-nm fi lament. This self-assembly mechanism establishes the nonpolarized nature of the intermediate fi lament, which is different from the polarized nature of microtubules and actin microfi laments (Hermann et al. 2007). Mature intermediate fi lament networks are highly strain resistant and exhibit strain hardening. Intermediate fi laments are classifi ed into six types based on similarities in amino acid sequence and protein structure. Different types of intermediate fi laments are expressed in tissues of specifi c differentiation patterns. Most intermediate fi lament proteins are keratins, classifi ed as either type I or type II, which are expressed in epithelia sheet tissues (Hanukoglu and Fuchs 1982). Simple keratins, K8/
K18, are the least specialized and likely the oldest keratin evolutionarily, whereas structural trichocyte hair keratins of the most recently evolved.
Type III and type IV groups, show some overlapping expression ranges and can be heteropolymeric in tissues; they are expressed in connective, nerve, muscle, and hematopoietic tissues. One of type III proteins, vimentin, represents minimal, nonepithelial intermediate filament expression and is characteristic of solitary cells. Type V proteins, the ubiquitous and ancient lamins, reinforce the nuclear envelope and interact with it via posttranslational modifi cations that produce membrane anchorage sites. Lamins, and probably all other intermediate fi lament proteins, are disassembled by phosphorylation during mitosis to allow chromosome separation and cytokinesis.
In multicellular organism, multiple intracellular signaling proteins can assemble into higher-order signaling machines that in some cases appear as helical architectures (Wu 2013). The formation and extension of the helical assemblies, also called signalosomes, transmits receptor activation information to cellular responses. During the process of the helical growth of signalosomes, infi nite assemblies provides the potential of signal amplifi cation by incorporating an over-stoichiometric number of signaling enzyme proteins into the complexes. In Drosophila, signalling mediator proteins, MyD88, IRAK4 and IRAK2 forms a specifi c signalosome called Myddosome complex, that assembled hierarchically into left-handed
helical oligomer of ~10 nm diameter. Formation of these Myddosome complexes brings the kinase domains of IRAKs into proximity to trigger phosphorylation and activation in signal transduction (Lin et al. 2010).
Helical symmetry may be especially suitable for regulating the pathway and the strength in signal transduction, because signalsomes with helical symmetries can evolve to accommodate a variable number of binding partners with specifi city. Helical assemblies may also reduce biological noise in signal transduction both kinetically and thermodynamically, because the growth of signalosome under helical symmetry requires cooperativity in the protein subunit association, which could generate stable signals above the background noise of stochastic molecular interactions.
2.2 Icosahedral and Conical Assembly
Different from the higher-order self-assembly of proteins native in cell, which often exhibits helical fi lament structures, viral proteins often self- assemble into more closed architectures like icosahedral and conical particles. The shell of a virus, called capsid, is composed of a number of non-covalently associated protein subunits and often exhibits higher-order symmetry including helical and icosahedral assemblies. The icosahedral architecture has 20 equilateral triangular faces (Branden and Tooze 1991).
The diameter of an icosahedral virus capsid is generally in the range of 50 to 150 nm. Each capsid face may consist of multiple protein subunits.
On the foot-and-mouth disease virus capsid, each face consists of three proteins named VP1–3. The icosahedral symmetry is quite common among viral capsids of different sizes. A regular icosahedron has 60 rotational symmetries and a symmetry order of 120 including the transformations that combine a rotation with a refl ection. Hence, given three asymmetric protein subunits on a triangular face of a regular icosahedron, 60 of such subunits are needed to assemble the icosahedron particle in an equivalent manner. Most viral capsid assemblies have more than 60 subunits and can be formed by more than one type of subunit. The variations in icosahedral capsid assemblies have been classifi ed on the basis of the quasi-equivalence principle, in which it is postulated that the protein subunits do not interact equivalently with one another, but nearly equivalently in an icosahedron assembly (Caspar and Klug 1962). An example is the Norwalk virus capsid, where there are 180 identical subunits forming an icosahedral architecture in a quasi-equivalent manner. There are 22 subgroups in icosahedral symmetry so that icosahedron architecture may be achieved via different protein assembly topology. In general, an icosahedral structure may be constructed from 12 pentamers (Fig. 2a and 2b). A number of hexamers could appear between pentamers in an icosahedron. The number of pentamers is fi xed but the number of hexamers can vary (Johnson and Speir 2009).
These icosahedrons can be constructed from pentamers and hexamers by minimizing the triangulation number, T, of nonequivalent locations that subunits occupy. The T-number adopts the particular integer values 1, 3, 4, 7, 12, 13, and so on, according to the formula T = h2 + k2 + hk, where h and k are nonnegative integers.
Although most known virus capsid assemblies adopt the icosahedron symmetry, there are exceptions such as the retroviruses. On the retrovirus capsids, a “fullerence cone” architecture may be formed by insertion of around 12 pentamers into a curved hexagonal lattice from around 200 hexamers, which closes the ovoid (Fig. 2c). Recent studies combining cryo-electron microscopy and molecular dynamics simulation suggested that the interfaces between neighboring hexamers and between pentamer and hexamers exhibit quasi-equivalence in the capsid lattice (Zhao et al.
2013). These phenomena imply that protein-protein assembly following the quasi-equivalence principle could be an effi cient, versatile approach for rational design of higher-order nanostructures.
2.3 Elastic Fiber Assembly
Elastic fibers represent intriguing biomaterials that are an integral component of the extracellular matrix in tissues (Muiznieks et al. 2010).
They are highly elastic and repetitively stretchable. Such properties are characteristic of vertebrate tissues whose physiological role requires a high degree of resilience over a lifetime. Elastic fi bers are found predominantly in connective and vascular tissue, lungs, and skin. 90% of the elastic fi ber consists of a protein called elastin, which is a polymer of the monomeric precursor tropoelastin and is the dominant contributor to fi ber elasticity. The self-assembly of elastin monomers into insoluble fi bers involves two steps.
Figure 2. Icosahedral and conical assembly. (a) The icosahedron assembly of Aquareovirus (EMD-5160) (Zhang et al. 2010). (b) The capsid of sulfolobus turreted icosahedral virus, with spikes distributed on the capsid surface. (PDB ID: 3J31) (Veesler et al. 2013). (c) The full atomic model of a complete conical HIV capsid (PDB ID: 3J3Y) (Zhao et al. 2013).
First, self-association of monomers occurs through hydrophobic domains in a process known as coacervation. Second, lysine residues of elastin are cross-linked by one or more members of the lysyl oxidase family. The non- elastin component of elastic fi bers is collectively referred to as microfi brils and includes fi brillins, fi bulins, and microfi bril-associated glycoproteins.
The peripheral microfi brils around elastin in elastic fi bers may contribute to elastin fi ber assembly as a guiding scaffold, as the microfi bril appears prior to elastin deposition. The elastin can elastically extend and contract in repetitive motion when hydrated. It has been suggested that elastin functions as an entropic spring. Upon relaxation from a stretching, elastic recoil is driven by increasing entropy that complies with increased disorder of the polypeptide chain and of surrounding water molecules. This implies certain degree of structural heterogeneity and disorder within the elastin monomer, where the formation of extended secondary structures is restricted in favor of transient and fl uctuating local motifs.
Water-swollen elastin fi bers are about 5–8 mm in diameter. Negative staining electron microscopy of elastin fi bers revealed distinct fi laments aligned parallel to the long axis of the fi ber (Gotte et al. 1974, Gotte et al.
1976). Similar fi laments were found in α-elastin, coacervates of tropoelastin, synthetic elastin-like polypeptides, fibrous elastin, and tropoelastin (Muiznieks et al. 2010 and references therein). These fi laments laterally associate into fi brils, which are bridged at regular intervals, and display a high propensity to aggregate into thick bundles (Pepe et al. 2007). Stretching of elastin fi bers reveals a transition from a glassy, plastic, disordered network to well-ordered fi laments aligned along the direction of the applied force.
The solution structure of individual hydrophobic domains of tropoelastin and elastin-derived peptides is disordered and fl exible in conformation. The fl uctuating β-turns within hydrophobic elastin-like sequences are believed to be labile and may contribute to elasticity. The elastin monomer is fl exible and highly dynamic. Structural disorder within monomer and aggregated elastin does not preclude the formation of preferred self-interactions or specifi c interactions with matrix and cell components, and thus the directional formation of fi bers. A high degree of combined composition of proline and glycine residues prevents the collapse of hydrophobic residues into compact globular structures by limiting the formation of backbone self- associations. Thus, the elastin monomer remains more hydrated and fl exible than globular proteins of similar size, where its conformational disorder plays an essential role in driving extensibility and elasticity.