Very rapid amyloid fibril formation by a bacterial lipase in the absence of a detectable lag phase.

The conversion of proteins from their soluble states into well-organized amyloid fibrils has received abundant attention. This process typically consists of three stages: lag, growth and plateau phases. In this study, the process of amyloid fibril formation by lipase from Pseudomonas sp. after diluting out urea was examined by Thioflavin T (ThT) fluorescence, Congo red (CR) binding, 8-anilinonaphthalene-1-sulfonic acid (ANS) binding, dynamic light scattering (DLS), circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, X-ray diffraction (XRD) and transmission electron microscopy (TEM). To exclude the presence of preformed aggregates in the pure lipase sample, aforementioned assays were also performed for the protein unfolded in urea before dilution. The aggregates formed immediately after dilution were found to bind to ThT and CR and contain a significant amount of β-sheet structure, as determined by far-UV CD and FTIR spectroscopies, as well as XRD analysis. Moreover, these aggregates present, at least in part, a fibrillar morphology, as deduced with TEM. This examination showed that lipase fibril formation proceeds quickly after dilution, within a few seconds, without a detectable lag phase. We also investigated bacterial inclusion bodies formed after expression of lipase in E. coli, providing evidence for the existence of rapidly formed amyloid-like structural and tinctorial properties in the lipase-containing inclusion bodies.