Timucin_2015_Biochemistry_54_3901

Thermoalkalophilic lipases are prone to aggregation from their dimer interface to which structural zinc is very closely located. Structural zinc sites have been shown to induce protein aggregation, but the interaction between zinc and aggregation tendency in thermoalkalophilic lipases remains elusive. Here we delineate the interplay between zinc and aggregation of the lipase from Bacillus thermocatenulatus (BTL2), which is taken to be a representative of thermoalkalophilic lipase. Results showed that zinc removal disrupted the BTL2 dimer, leading to monomer formation and reduced thermostability manifesting as a link between zinc and dimerization that leads to thermostability, while zinc addition induced aggregation. Biochemical and kinetic characterizations of zinc-induced aggregates showed that the aggregates obtained from the early and late stages of aggregation had differential characteristics. In the early stages, the aggregates were soluble and possessed native-like structures, while in the late stages, the aggregates became insoluble and showed fibrillar characteristics with binding affinities for Congo red and thioflavin T. The impact of temperature on zinc-induced aggregation was further investigated, and it was found that the native-like early aggregates could completely dissociate into functional lipase forms at high temperatures while dissociation of the late aggregates was limited. To this end, we report that the zinc-induced aggregation of BTL2 can be reversed by temperature switches and initiated by ordered aggregates in the early stages that gain fibrillar-like features over time. Insights revealed by this work contributes to the knowledge of aggregation mechanisms that exist in thermophilic proteins, reflecting the potential use of metal addition and/or removal to fine-tune aggregation tendency.