Paper Citing NAMD - Abstract
Huang, Jun; Jones, Bryan J.; Kazlauskas, Romas J.
Stabilization of an alpha/beta-Hydrolase by Introducing Pro line Residues: Salicylic Acid Binding Protein 2 from Tobacco
BIOCHEMISTRY, 54:4330-4341, JUL 21 2015
alpha/beta-Hydrolases are important enzymes for biocatalysis, but their stability often limits their application. We investigated a plant esterase, salicylic acid binding protein 2 (SABP2), as a Model alpha/beta-hydrolase. SABP2, shows typical stability to urea (unfolding free energy 6.9 +/- 1.5 kcal/mol) and to heat inactivation (T-1/2(15min) 49.2 +/- 0.5 degrees C). Denaturation in urea occurs in two steps, but heat inactivatibn occurs in a single Step. The first unfolding step in urea eliminates catalytic activity. Surprisingly, we found that the fitst unfolding likely corresponds to the unfolding of the larger catalytic domain. Replacing selected amino acid residues with proline atabilited SABP2. Proline restricts the flexibility of the unfolded protein, thereby shifting the equilibrium toward the folded conformation. Seven locations for proline substitution were chosen either by amino acid sequence alignment with a more stable homologue or by targeting flexible regions in SABP2. Introducing proline in the catalytic domain stabilized SABP2 to the first unfolding in urea for three of five cases: L46P (+0.2 M urea), S70P (+0.1), and E215P (+0.9). Introducing,proline in the cap domain clid not stabilize SABP2 (two of two cases), supporting the assignment that the first unfolding corresponds to the catalytic domain. Proline substitutions in both domains stabilized SABP2 to heat inactivation: L46P (Delta T-1/2(15min) = +6.4 degrees C), S70P (+5.4), S115P (+1.8), S141P (+4.9), and E215P (+4.2). Combining substitutions did not further 'increase the stability to urea -denaturation, but -dramatically increased resistance to heat inactivation: L46P-S70P Delta T-1/2(15min) = +25.7 degrees C. This straightforward proline substitution approach may also atabilite other alpha/beta-hydrolases.
