Encoding Structure in Intrinsically Disordered Protein Biomaterials

Abstract

In nature, proteins range from those with highly ordered secondary and tertiary structures to those that completely lack a well-defined three-dimensional structure, termed intrinsically disordered proteins (IDPs). IDPs are generally characterized by one or more segments that have a compositional bias toward small hydrophilic amino acids and proline residues that promote structural disorder and are called intrinsically disordered regions (IDRs). The combination of IDRs with ordered regions and the interactions between the two determine the phase behavior, structure, and function of IDPs. Nature also diversifies the structure of proteins and thereby their functions by hybridization of the proteins with other moieties such as glycans and lipids; for instance, post-translationally glycosylated and lipidated proteins are important cell membrane components. Additionally, diversity in protein structure and function is achieved in nature through cross-linking proteins within themselves or with other domains to create various topologies. For example, an essential characteristic of the extracellular matrix (ECM) is the cross-linking of its network components, including proteins such as collagen and elastin, as well as polysaccharides such as hyaluronic acid (HA). Inspired by nature, synthetic IDP (SynIDP)-based biomaterials can be designed by employing similar strategies with the goal of introducing structural diversity and hence unique physiochemical properties. This Account describes such materials produced over the past decade and following one or more of the following approaches: (1) incorporating highly ordered domains into SynIDPs, (2) conjugating SynIDPs to other moieties through either genetically encoded post-translational modification or chemical conjugation, and (3) engineering the topology of SynIDPs via chemical modification. These approaches introduce modifications to the primary structure of SynIDPs, which are then translated to unique three-dimensional secondary and tertiary structures. Beginning with completely disordered SynIDPs as the point of origin, structure may be introduced into SynIDPs by each of these three unique approaches individually along orthogonal axes or by combinations of the three, enabling bioinspired designs to theoretically span the entire range of three-dimensional structural possibilities. Furthermore, the resultant structures span a wide range of length scales, from nano- to meso- to micro- and even macrostructures. In this Account, emphasis is placed on the physiochemical properties and structural features of the described materials. Conjugates of SynIDPs to synthetic polymers and materials achieved by simple mixing of components are outside the scope of this Account. Related biomedical applications are described briefly. Finally, we note future directions for the design of functional SynIDP-based biomaterials.

Figure 1:. Sequence and LLPS behavior of different types of SynIDPs.

A. ELPs undergo an LCST phase transition, which can be controlled by ELP length (n) and the identity of the guest residue (X). B. RLPs undergo a UCST phase transition which can be controlled by RLP length (n) and the specific identity of the amino acids in the octapeptide repeat unit. Colored (not grey) amino acids in panel B can be replaced by other amino acids classified in the same group. Classification of amino acids: S-N/Q/T are polar, uncharged amino acids (marked in blue); R-K are positively charged amino acids (marked in purple), D-E are negatively charged amino acids (marked in green); G and P are structure breaking amino acids (marked in grey) and the remaining amino acids are hydrophobic (marked in red).

Figure 2:. Incorporation of ordered domains into SynIDPs.

A. POPs as an example of helices (oligoalanines) incorporated into a SynIDP (ELP). POPs self-assemble into solid-like, microporous fractal networks upon heating above their T_t, as evidenced by structured illumination microscopy. Adapted with permission from ref. . Copyright 2018, Nature Research. B. SELPs as an example of β-strands (silk-like) incorporated into a SynIDP (ELP). SELPs self-assemble into fibers, as evidenced by atomic force microscopy. Adapted with permission from ref. . Copyright 2019, American Chemical Society. C. ZS-EI-ELR as an example of both helices (leucine zippers) and β-strands (silk-like) incorporated into a SynIDP (ELP). ZS-EI-ELR forms a hydrogel, driven by the thermal phase transition of the ELP domain and stabilized by the β-sheets formed by the crystallizable silk-like domain and coiled coil formed by the leucine zippers.

Figure 3:. Incorporation of lipids into SynIDPs.

M-ELPs were the first example of exploiting a PTM—N-myristoylation—to append a lipid to an ELP to create a genetically encoded hybrid biomaterial. M-ELPs self-assemble into nanoscale micelles, driven by the amphiphilicity of the construct conferred by the hydrophobicity of the appended lipid. The size and shape of M-ELPs may be tuned by adjusting the ELP length.

Figure 4:. Topology engineering of SynIDPs.

A. Demonstration of using pAzF (purple circles) to create stable mesoparticles. PCE and PCD are mixed, and the temperature is raised above the T_t of PCE but below the T_t of PCD. PCD acts as a surfactant to stabilize PCE particles. UV-irradiation covalently crosslinks the core. Data from ref. . B. Demonstration of SpyTag/SpyCatcher system to create nonlinear ELP topologies. Placing SpyTag and SpyCatcher on the same ELP chain leads to cyclization of the ELP. Positioning the SpyTag and SpyCatcher on different ELP chains leads to the formation of 3-armed proteins. Data from ref. . C. ELP dendrimers are synthesized using a divergent strategy with Fmoc-based SPPS. Lysines are used as branching points. Data from ref. .

Figure 5:. Incorporating ordered protein domains and non-protein moieties.

FAMEs are composed of an ELP, a non-protein moiety (myristoyl lipid group), and an ordered protein domain (structure-directing peptide). The hierarchical self-assembly of FAMEs is driven by the hydrophobicity of the myristoyl group and the presence of the β-sheet-forming peptide, as well as the thermal responsiveness of the ELP. FAMEs exhibit a range of structures during the three-stage self-assembly process, from cylindrical micelle morphology (visualized by cryogenic transmission electron microscopy), to liquid-like coacervates and finally to macroscopic bundled fibers (both visualized by spinning disk confocal laser microscopy). Adapted with permission from ref. . Copyright 2018 Nature Research.