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pH‐dependent aggregation of cutinase is efficiently suppressed by 1,8‐ANS
Author(s) -
Pedersen Shona,
Nesgaard Lise,
Baptista Ricardo P.,
Melo Eduardo P.,
Kristensen Søren R.,
Otzen Daniel E.
Publication year - 2006
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.20598
Subject(s) - cutinase , chemistry , lysozyme , hydrophobic effect , lipase , molecule , stereochemistry , osmolyte , enzyme , crystallography , biophysics , organic chemistry , biochemistry , biology
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 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com