Abstract
We have studied the thermal stability of the triglyceride‐hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 μ__M__ decreases the apparent melting temperature by 19°C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes, the hydrophobic fluorophore 1,8‐ANS completely and saturably suppresses this effect, restoring 70% of enzymatic activity upon cooling. ANS binds strongly to native cutinase as a noncompetitive inhibitor with up to 5 ANS per cutinase molecule. Only the first ANS molecule stabilizes cutinase; however, the last 4 ANS molecules decrease T~m~ by up to 7°C. Similar pI‐dependent aggregation and suppression by ANS is observed for T. lanuginosus lipase, but not for lysozyme or porcine α‐amylase, suggesting that this behavior is most prevalent for proteins with affinity for hydrophobic substrates and consequent exposure of hydrophobic patches. Aggregation may be promoted by a fluctuating ensemble of native‐like states associating via intermolecular β‐sheet rich structures unless blocked by ANS. Our data highlight the chaperone activity of small molecules with affinity for hydrophobic surfaces and their potential application as stabilizers at appropriate stoichiometries. © 2006 Wiley Periodicals, Inc. Biopolymers 83: 619–629, 2006
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