Thermostability at different incubation concentrations.

<p>Residual lipase activity after thermal incubation for 30 minutes at concentrations of (A) 1 µM and (B) 50 µM. Student's t-test were performed to determine the significant differences in thermostability of W211A with respect to BTL2 (*p=0.1 and **p=0.05). </p

The structural impact of W211A mutation on the subunit interface.

<p>The subunit interface is rendered by van der Waals surface using the colors; green for chain A, blue for chain B, and <i>red</i> for 211 (W or A) that is found in chain B. The arrows indicate the residues that change conformation upon W211A mutation in (A) chain A and (B) chain B. The representation in (A) has been rotated 180° around the left diagonal axis to obtain (B), and the snapshots were taken from the simulations performed at 75°C.</p

Crystal structure of the dimer BTL2.

<p>Two subunits, chain A and B are shown in black and silver, respectively, where the lids are colored in orange for both. The subunit interface is rendered by van der Waals surface: blue for chain A and <i>red</i> for chain B. The lid tryptophans W211 and W234 are shown in green sticks. (B) The aromatic cluster and (C) the network of hydrogen bonds were shown in stick models (C: cyan, N: blue, O: red).</p

W211 in the active conformation.

<p>The domain formed by W211 in the open-active conformation (PDB ID: 2W22) is rendered by van der Waals surface. H87-G88 (green) and P232-V233-S236 (yellow) tightly packs the side chain of W211 colored in (red). </p

The ANS fluorescence at 460 nm.

<p>The closed circles show the fluorescence from the lipases in 5 mM Tris-Cl at pH 7.0 and the open circles show the effect of 2-propanol. </p

The Conserved Lid Tryptophan, W211, Potentiates Thermostability and Thermoactivity in Bacterial Thermoalkalophilic Lipases

<div><p>We hypothesize that aggregation of thermoalkalophilic lipases could be a thermostability mechanism. The conserved tryptophans (W211, W234) in the lid are of particular interest owing to their previous involvements in aggregation and thermostability mechanisms in many other proteins. The thermoalkalophilic lipase from <i>Bacillus thermocatenulatus</i> (BTL2) and its mutants (W211A, W234A) were expressed and purified to homogeneity. We found that, when aggregated, BTL2 is more thermostable than its non-aggregating form, showing that aggregation potentiates thermostability in the thermoalkalophilic lipase. Among the two lid mutants, the W211A lowered aggregation tendency drastically and resulted in a much less thermostable variant of BTL2, which indicated that W211 stabilizes the intermolecular interactions in BTL2 aggregates. Further thermoactivity and CD spectroscopy analyses showed that W211A also led to a strong decrease in the optimal and the melting temperature of BTL2, implying stabilization by W211 also to the intramolecular interactions. The other lid mutant W234A had no effects on these properties. Finally, we analyzed the molecular basis of these experimental findings <i>in-silico</i> using the dimer (PDB ID: 1KU0) and the monomer (PDB ID: 2W22) lipase structures. The computational analyses confirmed that W211 stabilized the intermolecular interactions in the dimer lipase and it is critical to the stability of the monomer lipase. Explicitly W211 confers stability to the dimer and the monomer lipase through distinct aromatic interactions with Y273-Y282 and H87-P232 respectively. The insights revealed by this work shed light not only on the mechanism of thermostability and its relation to aggregation but also on the particular role of the conserved lid tryptophan in the thermoalkalophilic lipases.</p> </div