Abstract
Capsular antigen fragment 1 (Caf1) is an oligomeric protein consisting of 15 kDa monomeric subunits that are non-covalently linked through exceptionally strong and kinetically inert interactions into a linear polymer chain. It has been shown that after its thermal depolymerisation into unfolded monomeric subunits, Caf1 is able to efficiently repolymerise in vitro to reform its polymeric structure. However, little is known about the nature of the repolymerisation process. An improved understanding of this process will lead to the development of methods to better control the lengths of the repolymerised species, and ultimately, to better design of the properties of Caf1-based materials. Here we utilize small-angle X-ray scattering to estimate the size of Caf1 polymers during the first 24 h of the re-polymerisation process. Analytical ultracentrifugation measurements were also used to investigate the process post-24 h, where the rate of repolymerisation becomes considerably slower. Results show that in vitro polymerisation proceeds in a linear manner with no evidence observed for the formation of a lateral polymer network or uncontrolled aggregates. The rate of Caf1 in vitro repolymerisation was found to be concentration-dependent. Importantly, the rate of polymer growth was found to be relatively fast over the first few hours, before continuing at a dramatically slower rate. This observation is not consistent with the previously proposed step-growth mechanism of in vitro polymerisation of Caf1, where a linear increase in polymer length would be expected with time. We speculate how our observations may support the idea that the polymerisation process may be occurring at the ends of the chains with monomers adding sequentially. Our findings will contribute towards the development of new biomaterials for 3D cell culture and bio-printing.
Highlights
Capsular antigen fragment 1 (Caf1) is an oligomeric protein consisting of 15 kDa monomer subunits that are non-covalently linked into a linear polymer chain (Fig. 1a)
The early stages of Caf1 repolymerisation can be observed by small-angle X-ray scattering (SAXS)
The q × I(q) estimate for a hypothetical Gaussian coil simulating scattering of disordered Caf1 monomer (Rg = 41.3 Å, 5 mg/ mL) at zero time was made to show the relatively low contribution that results from the disordered monomer (Fig. 2c)
Summary
Capsular antigen fragment 1 (Caf1) is an oligomeric protein consisting of 15 kDa monomer subunits that are non-covalently linked into a linear polymer chain (Fig. 1a). We speculate that heat perturbs the tertiary structure of the acceptor to the extent that it is no longer complementary to the N-terminal donor strand, resulting in decomplexation This thermal unfolding-depolymerisation process is reversible, and upon cooling the subunits were shown to refold with concomitant recomplexation of the N-terminal donor strand, reforming the polymer chain (Fig. 1c). Caf1’s capacity for reversible thermal unfolding is maintained even when extensively cross-linked to form a hydrogel network and endows their hydrogels with the capacity to melt and reform, i.e., undergo a reversible gel–sol transition This feature of Caf presents new material possibilities, and we were able to successfully demonstrate hydrogel reshaping, welding and blending. It was even possible to encapsulate live cells inside the hydrogel network, avoiding the use of reactive chemical cross linkers or expensive biorthogonal chemistries (Dura et al 2020)
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