Human Collagen
Human collagen is a complex protein to produce recombinantly. The tight left-handed helix structure is facilitated by numerous hydroxyproline residues which must be produced by post-translational modification by an enzyme with complex quaternary structure only naturally found in a single relatively common yeast strain, Hansenula polymorpha. Hydroxyproline is also not a valid candidate for use of an orthogonal tRNA system in an amberless cell, because the prolyl-tRNA synthetase is sufficiently promiscuous to allow formation of hydroxyprolyl-tRNA in substantial quantities as well. The traditional collagen motif id Gly-X-Y, with X often representing proline and Y hydroxyproline; these relative locations in the helix are essential for their contributions to its formation and so randomized inclusion of both could actually be counterproductive. Even working in H. polymorpha, which in addition to not being cultured in most labs has finnicky codon preferences, there are a number of problems. Collagen genes themselves are ill-suited to genetic manipulation, with glycine and proline codons (GGN and CCN respectively) making up such a large proportion of the sequence, both very high GC content and potential self complementarity are real concerns in addition to general repetitive sequence issues. Even after transformation and successful translation, human collagen undergoes post-translational glycosylation and cleavage before forming a number of different heterotrimers, with cross links depending on specific positioning of a number of different post-translationally modified amino acids. All this is not to say that recombinant synthesis of human collagen is impossible; it has in fact been done. However, replicating that work would have been quite difficult in the time available without representing substantial innovation. Instead, we looked to possible mimetics.Bacterial Collagen
Fortunately for what was rapidly becoming an Escherichia coli-centric team, there exists a whole class of proteins known as "bacterial collagens". These proteins have the same supersecondary structure and Gly-X-Y repeat as human collagen without relying on hydroxyproline in the Y position, which makes these sequences easier to manipulate and bacterial synthesis possible. Unfortunately, despite the suggestive moniker, bacterial collagen has not been well adapted to provide structural integrity to large regions of extracellular matrix in areas of regular high stress such as arterial walls and skin. As such, it has no natural mechanism to form a network of long, cross-linked fibers, and there is a dearth of data on how such a matrix might perform. Still, it might prove a viable collagen mimetic given successful assembly of a fibrous matrix. Returning to humman collagen mimetics for a moment, the length of native collagen has been one factor making production difficult, and there has been some interesting work done with relatively short peptides working around this difficulty.Peptide Tessellation
Some recent work has been done to create a potentially infinitely long self-assembling collagen-like triple helix, made possible by the design and synthesis of short peptides which assemble in a staggered fashion into a triple helix with sticky ends, permitting the continued addition of the peptide monomer indefinitely into a long fiber. Sticky ends are most familiar in DNA applications such as restriction cloning, but sticky-ended DNA fragments can also be created by combining two DNA single strands (as in the work of this team on melanin binding) due to the specificity of base pair interactions. Tanrikulu et al. used lysine/aspartate charge pairs to similar effect, forcing a staggered assembly. Cejas et al. chose to use short 30-residue length triple helices which would interact and form longer chains through stacking interactions of aromatic terminal residues. These are just two examples of what is a fairly diverse field of collagen-mimetic peptide exploration, but what they share is a dependence on peptide synthesis. A transition to recombinant synthesis in an organism is difficultThese short, non-native structures would not necessarily be post-translationally modified correctly and would be difficult to purify, with even short purification tags resulting in major disruption of the structure and even an initial formyl-methionine potentially destructive. For much the same reason, they are difficult to modify further, and while the work of a number of authors has shown the potential for peptide-based models to interact with human cells, the lack of any cross-linking means that they may not be stable under physical stress, an essential feature of any membrane material. Tanrikulu and Raines may have developed a cysteine-homocysteine system for intra-strand disulfide cross-links, but this too is best-suited for solid-phase peptide synthesis. Our approach works within a bacterial production system.Collagen, Keratin, and Elastin (Oh my)
The bacterially produced collagen mimetic we designed draws from a few additional sources, wading into the uncertain waters of rational design in the process. Yoshizumi et al. were able to demonstrate the utility of a designed coiled-coil homotrimerization domain in assisting the folding of a bacterial collagen domain. Coiled-coil domains, the most famous example of which forms the fundamental dimer structure of keratin, are defined by a heptad repeat structure and can govern the association of anywhere between two and seven proteins; the example here was desigend to form homotrimers which retained their structure above 90 °C. The authors evaluate the effect of different locations of this domain on the refolding of a single bacterial collagen domain, concluding that a single coiled-coil domain at the N-terminus is optimal for the most rapid and complete refolding. We created a close analog to this construct (modified for convenient PCR amplification) with a purification tag and submitted it as BBa_K2027007.This general approach might be extended to different coiled-coil and target proteins; in particular, the potential applications of an effective heterotrimerization domain are even more expansive. A candidate coiled-coil obligate heterotrimer with a defined orientation was designed in 1995 by Nautiyal et al., with all homotrimers and two-component heterotrimers made unfavorable by charge pair interactions. Full heterotrimers have a higher melting point (apparent Tm = 87.5 °C) than the next-most stable arrangement by at least 15 °C, potentially allowing thermal cycling to increase specificity. A first step toward using this domain would be attempting to replace the coiled-coil in Yoshizumi et al.'s device with the coiled-coil domains making up the heterotrimer; bricks BBa_K2027044, BBa_K2027037, and BBa_K2027038 represent these constructs. Observing under what conditions, if any, specifity is maintained in the presence of a weaker homotrimerization domain (the bacterial collagen) could give some indication of the utility of these domains in organizing proteins. Collagen-like triple helices generally have melting temperatures below 40 °C, so the coiled-coil domains have a clear thermodynamic advantage and at least one has been shown to nucleate trimerization of an unfolded bacterial collagen trimer, but the kinetics of initial formation of trimers was not studied and in fact could not have been studied in the setting of the first construct. Instead, the bacterial collagen domain was unfolded while leaving the coiled-coil trimers intact; it remains possible that at room temperature, interaction of bacterial collagen domains dominates the trimerization process. The relative stability of the heterotrimer will still allow for formation of the desired heterotrimeric construct at an elevated temperature regardless of room temperature kinetics, but it would be interesting to study the kinetics nonetheless. Still, all constructs so far are short, blunt-ended fibers incapable of assembling into a matrix. However, the modular nature of this assembly suggests the possibility of large-scale cross-linking in a way peptide tesselation models might have more difficulty supporting.
Cross-linking is essential for proper functioning of collagen in a structural context; covalent linkages ensure not just thermal but also mechanical stability of quaternary protein associations. Bacterial collagen offers no help here; human collagen is replete with intra- and inter-strand cross-links but they depend on precise positioning and the action of multiple mammalian enzymes. Fortunately, a simple protein fiber cross-linking module has already had its efficacy demonstrated: exons 21 and 23 from human tropoelastin have been shown to cross-link coacervated elastin-like monomers in the presence of a lysine-oxidizing agent. The single relavant enzyme is not even necessary; as is shown in the PQQ aptamer purification project, it might soon be enough to purchase a cofactor supplement at a local drug store and add copper sulfate. To explore this, another set of constructs was created with this cross-linking domain at the N-terminus. These are bricks BBa_K2027003, BBa_K2027004, BBa_K2027005, and BBa_K2027006. These constructs need to be studied to evaluate the effect of the addition on trimerization and the potential for cross-linking. Even if cross-linking is successful, these would still be short, blunt fibers, but governance of their assembly by formation of a heterotrimer opens the door to sticky end use.
Parts BBa_K2027002 and BBa_K2027045 are the conjugations of the three heterotrimer parts (with and without cross-linking domains, respectively). Part BBa_K2027002 is represented in both detailed and simplified schematics in Figure N. Due to the directionality and specificity of trimer formation, the preferred assembly has sticky ends leaving the possibility of substantially longer fibers such as that shown in simplified form in Figure NN.
