Title:Impact of Salt Concentration and pH on Surface Charged Residues: Controlling Protein Association Pathways in Carboxylesterase EstGtA2
Volume: 24
Issue: 6
Author(s): Jessica Kelly Moisan, Fatma Meddeb-Mouelhi, David M. Charbonneau and Marc Beauregard*
Affiliation:
- CRML, Université du Québec à Trois-Rivières, C.P. 500, Trois-Rivières (Québec),Canada
Keywords:
Thermal denaturation, circular dichroism, dynamic light scattering, protein aggregation, protein association, Est-
GtA2.
Abstract: Background: Understanding the relationship between enzymatic stability and the amino
acid sequence encoding carboxylesterases is of utmost importance.
Objectives: Here we thoroughly characterized the behavior of the carboxylesterase EstGtA2 from
Geobacillus thermodenitrificans during thermal denaturation at different pH with various salt concentrations.
Method: EstGtA2 was characterized by circular dichroism regarding conformation and thermal stability,
by dynamic light scattering for detection of association/aggregation, by enzymatic assays for
activity and by monitoring the impact of heat treatments on activity.
Results: Our investigation revealed a particular dependence between aggregation/association and
preservation of secondary structures upon heating in EstGtA2. At pH 7, 8 and 9, depending on salt
concentration, a folded but non-native associated state characterised by an apparent particle size of
300 nm resisted secondary structure unfolding up to 95°C.
Conclusion: The paths leading to various aggregative states were found to be controlled by pH (depending
on proximity to pI) and to a lesser extent, ionic strength, suggesting that ionic interactions at
the surface of the protein are responsible for behavior of EstGtA2. The various paths available to
EstGtA2 could be important for protection of
Geobacillus termodenitrificans when exposed to heat
stress. The understanding and/or control of these paths would allow for optimal use of EstGtA2 in
industrial processes.
