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

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. [ABSTRACT FROM AUTHOR]