Insight into the aggregation of lipase from Pseudomonas sp. using mutagenesis: protection of aggregation prone region by adoption of α-helix structure

Abstract

Previously, a lipase purified from a Pseudomonas source showed to form amyloid fibril structure very rapidly in the absence of a detectable lag phase. In this process the urea-unfolded enzyme encounters a medium close to physiological, but is unable to fold and, therefore, the main driving force of aggregation lies in the sequence of the protein and in its aggregation-promoting regions (APRs). Two regions with the highest propensity to aggregate were identified. These were Regions 51-57 and 160-172 as they were found with all four prediction algorithms. Two mutants of lipase, F171E and I52E, were selected and their propensity to aggregate was evaluated using thioflavin T (ThT), Congo red binding, circular dichroism, transmission electron microscopy (TEM) and dynamic light scattering. While I52E lipase formed aggregates that were capable of amyloid dye binding, showed a typical β-sheet structure and amorphous/fibrillar morphology, the aggregates formed by the F171E mutant indicated diminished ThT binding, lower light scattering, a smaller content of β-sheet structure and a lower presence of aggregates by TEM imaging. These data indicate that the region of the Sequence 160-172 is an APR region of this protein and lead to the suggestion of strategies aimed at promoting the solubility of this protein.

Keywords: amyloid prone region; lipase; protein engineering; protein misfolding.