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Probing the oligomeric re-assembling of bacterial fimbriae in vitro: a small-angle X-ray scattering and analytical ultracentrifugation study.

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.

SUBMITTER: Solovyova AS

PROVIDER: S-EPMC8190007 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Magnetosomes are membrane-enveloped single-domain ferromagnetic nanoparticles enabling the navigation of magnetotactic bacteria along magnetic field lines. Strict control over each step of biomineralization generates particles of high crystallinity, strong magnetization, and remarkable uniformity in size and shape, which is particularly interesting for many biomedical and biotechnological applications. However, to understand the physicochemical processes involved in magnetite biomineralization, close and precise monitoring of particle production is required. Commonly used techniques, such as transmission electron microscopy (TEM) or Fe measurements, allow only for semiquantitative assessment of the magnetosome formation without routinely revealing quantitative structural information. In this study, lab-based small-angle X-ray scattering (SAXS) is explored as a means to monitor the different stages of magnetosome biogenesis in the model organism Magnetospirillum gryphiswaldense SAXS is evaluated as a quantitative stand-alone technique to analyze the size, shape, and arrangement of magnetosomes in cells cultivated under different growth conditions. By applying a simple and robust fitting procedure based on spheres aligned in linear chains, it is demonstrated that the SAXS data sets contain information on both the diameter of the inorganic crystal and the protein-rich magnetosome membrane. The analyses corroborate a narrow particle size distribution with an overall magnetosome radius of 19 nm in Magnetospirillum gryphiswaldense Furthermore, the averaged distance between individual magnetosomes is determined, revealing a chain-like particle arrangement with a center-to-center distance of 53 nm. Overall, these data demonstrate that SAXS can be used as a novel stand-alone technique allowing for the at-line monitoring of magnetosome biosynthesis, thereby providing accurate information on the particle nanostructure.IMPORTANCE This study explores lab-based small-angle X-ray scattering (SAXS) as a novel quantitative stand-alone technique to monitor the size, shape, and arrangement of magnetosomes during different stages of particle biogenesis in the model organism Magnetospirillum gryphiswaldense The SAXS data sets contain volume-averaged, statistically accurate information on both the diameter of the inorganic nanocrystal and the enveloping protein-rich magnetosome membrane. As a robust and nondestructive in situ technique, SAXS can provide new insights into the physicochemical steps involved in the biosynthesis of magnetosome nanoparticles as well as their assembly into well-ordered chains. The proposed fit model can easily be adapted to account for different particle shapes and arrangements produced by other strains of magnetotactic bacteria, thus rendering SAXS a highly versatile method.

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Probing the oligomeric re-assembling of bacterial fimbriae in vitro: a small-angle X-ray scattering and analytical ultracentrifugation study.

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