Part:BBa_K2027038:Design
Bacterial Collagen with Coiled-Coil Heterotrimerization Domain 3
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 75
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 519
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 72
Illegal BsaI.rc site found at 65
Design Notes
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.1 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 four proteins fairly easily;2 the example here was designed to form homotrimers which retained their structure above 90 °C.1 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.1 We created a close analog to this construct (modified for convenient PCR amplification) with a purification tag and submitted it as Part: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.2 Full heterotrimers have a higher melting point (apparent Tm = 87.5 °C) than the next-most stable arrangement by at least 15 °C,2 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 Part:BBa_K2027044, Part:BBa_K2027037, and Part:BBa_K2027038 represent these constructs. Observing under what conditions, if any, specificity 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;1 it remains possible that at room temperature, interaction of bacterial collagen domains dominates the trimerization process. The relative stability of the heterotrimer will 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.3 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.4 The single relavant enzyme is not even necessary; as is shown in the PQQ aptamer purification[http://2016.igem.org/Team:Stanford-Brown/SB16_BioMembrane_AptamerPurification] 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 the relevant bricks: Part:BBa_K2027003, Part:BBa_K2027004, Part:BBa_K2027005, and Part: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 the short, blunt fibers, but governance of their assembly by formation of a heterotrimer opens the door to sticky end use.
Below is the forward and reverse sequencing verification for the part, showing that the digestion left the heterotrimeric coiled coil and bacterial collagen domain intact.
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