Influence of Centrifugation and Shaking on the Self-Assembly of Lysozyme Fibrils

Abstract

Protein self-assembly into fibrils and oligomers plays a key role in the etiology of degenerative diseases. Several pathways for this self-assembly process have been described and shown to result in different types and ratios of final assemblies, therewith defining the effective physiological response. Known factors that influence assembly pathways are chemical conditions and the presence or lack of agitation. However, in natural and industrial systems, proteins are exposed to a sequence of different and often complex mass transfers. In this paper, we compare the effect of two fundamentally different mass transfer processes on the fibrilization process. Aggregation-prone solutions of hen egg white lysozyme were subjected to predominantly non-advective mass transfer by employing centrifugation and to advective mass transport represented by orbital shaking. In both cases, fibrilization was triggered, while in quiescent only oligomers were formed. The fibrils obtained by shaking compared to fibrils obtained through centrifugation were shorter, thicker, and more rigid. They had rod-like protofibrils as building blocks and a significantly higher β-sheet content was observed. In contrast, fibrils from centrifugation were more flexible and braided. They consisted of intertwined filaments and had low β-sheet content at the expense of random coil. To the best of our knowledge, this is the first evidence of a fibrilization pathway selectivity, with the fibrilization route determined by the mass transfer and mixing configuration (shaking versus centrifugation). This selectivity can be potentially employed for directed protein fibrilization.

Robust assembly of the aldehyde dehydrogenase Ald4p in Saccharomyces cerevisiae

“…In this study, the way we prepared the yeast samples for imaging was different from our previous studies with other filament-forming enzymes. Similar to a recent study ( Krzek et al, 2022 ), we noticed that the filament length of Ald4p-GFP is sensitive to shaking conditions (unpublished data). In order to circumvent this problem, we scraped the cells directly from the agar plate, resuspended them in 1× PBS, prepared a wet slide, and imaged the filaments by fluorescence microscopy (the whole process was limited to 30 min for each prep).…”

Automated microscopic measurement of fibrinaloid microclots and their degradation by nattokinase, the main natto protease

“…This was then followed by immediately pipetting 15 μL of assay sample on 15-well slide 'angiogenesis' glass bottom plate used without a lid (I https://ibidi.com/chambered-coverslips/245--slide-15-well-3d-glass-bottom.html), reproduced in Figure 3, and wit shaking (cf. [142,[297][298][299][300][301][302][303][304][305][306][307][308][309]). The excitation wavelength band for ThT was set at 450 nm to 499 nm and the emissio 499 nm to 529 nm.…”