Artificial Protein Block Copolymers Blocks Comprising Two Distinct Self-Assembling Domains

Synthetic block copolymers comprising two or more compositionally distinct chains have attracted significant attention due to their ability to self-assemble into ordered microstructures. Although tremendous progress has been made in the chemical synthesis of polymers, the unsurpassed degree of control and diversity of monomers combined with advances in recombinant DNA technology permits the synthesis of unique artificial protein-derived block polymers. These include silk–elastin, elastin–elastin hybrids of varying elastin blocks, and helix–random coil–helix triblock combinations. These polymers consist of nearly similar self-assembling chains, as in the case of silk–elastin and elastin–elastin hybrids, or one self-assembling motif fused to a disordered random motif. Herein, we describe three block copolymers comprising two distinct self-assembling chains—elastin (E) and cartilage oligomeric matrix protein coiled-coil (COMPcc; C)—fused in two orientations (EC and CE) and a final construct in which an additional E block is appended (ECE; Figure 1A–C). Remarkably, the polymer structures as well as temperature and small-moleculedependent assembly rely on the block orientation and the number of blocks. Elastins consist of pentapeptide (VPGXG)n repeating units, where X is an interchangeable amino acid, that self-assemble into helical b-spirals. 7] Elastins exhibit a lower critical solution temperature (Tt) behaviour that can be tuned by varying the identity of X and the number of repeats (n). By contrast, COMPcc self-assembles into a homopentamer of parallel a-helical coiled-coils to produce a hydrophobic pore that is 7.3 nm long with a diameter of 0.2–0.6 nm. Individually, the E and C domains exhibit unique modes of self-assembly and conformation; E undergoes phase separation while C can bind small molecules. Each polymer consists of compositionally identical E and C motifs into which a short A2(TA)n spacer is incorporated at the juncture between the domains to ensure that each block is able to self-assemble as required (Figure 1A–C). A critical feature of smart biomaterials is the ability of the polymers to self-assemble as a function of environmental cues such as pH and ionic strength. Previous studies have shown that elastin and coiled-coil domains can be influenced by pH and salt conditions. The synthetic versatility of these block polymer constructs permits the exploration of how the orientation and the number of blocks influence their physicochemical properties. All block polymers have been overexpressed, purified and characterised. The molar masses of EC, CE and ECE are 22731, 22911 and 35188 Da, respectively. Although SDSPAGE analysis of the purified polymers reveals a slightly higher molecular weight for EC, CE and ECE, due to the E portion of the block polymers (Figure 1D), the exact masses were confirmed by MALDI. To determine the conformations of the block polymers, far-UV circular dichroism (CD) measurements were conducted. The homopolymer C adopted a helical structure that exhibited a transition to random configuration as the temperature was raised. In contrast, E adopted an initial b-turn that loses its structure at higher temperatures. Although nearly identical in composition, the EC and CE diblocks differed in secondary structure and exhibited distinct temperature-dependent conformational changes (Figure 2A, B). In the case of Figure 1. Amino acid sequences and structures of A) EC, B) CE and C) ECE. D) SDS-PAGE identifying protein fusions. E) Vitamin D3.