Urea titration of a lipase from Pseudomonas sp. reveals four different conformational states, with a stable partially folded state explaining its high aggregation propensity.

The conversion of soluble proteins into amyloid fibrils has importance in protein chemistry, biology, biotechnology and medicine. A novel lipase from Pseudomonas sp. was previously shown to have an extremely high aggregation propensity. It was therefore herein studied to elucidate the physicochemical and structural determinants of this extreme behaviour. Amyloid-like structures were found to form in samples up to 2.5-3.0 M using Thioflavin T fluorescence and Congo red binding assays. However, dynamic light scattering (DLS), static light scattering and turbidimetry revealed the existence of aggregates up to 4.0 M urea, without amyloid-like structure. Two monomeric conformational states were detected with intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonate (ANS) binding and circular dichroism. These were further characterized in 7.5 M and 4.5 M urea using enzymatic activity measurements, tryptophan fluorescence quenching, DLS and nuclear magnetic resonance (NMR) and were found to consist of a largely disordered and a partially folded state, respectively, with the latter appearing stable, cooperative, fairly compact, non-active, α-helical, with largely buried hydrophobic residues. The persistence of a stable structure up to high concentrations of urea, in the absence of sequence characteristics typical of a high intrinsic aggregation propensity, explains the high tendency of this enzyme to form amyloid-like structures.