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Involvement of Cysteine Residues and Domain Interactions in the Reversible Unfolding of Lipoxygenase-1

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JBC-05-99
 
Title Involvement of Cysteine Residues and Domain Interactions in the Reversible Unfolding of Lipoxygenase-1
 
Creator Sudharshan, E
Appu Rao, A. G.
 
Subject 16 Enzyme Chemistry
 
Description Urea-induced unfolding of lipoxygenase-1 (LOX1) at
pH 7.0 was followed by enzyme activity, spectroscopic
measurements, and limited proteolysis experiments.
Complete unfolding of LOX1 in 9 M urea in the presence
of thiol reducing or thiol modifying reagents was observed.
The aggregation and oxidative reactions prevented
the reversible unfolding of the molecule. The loss
of enzyme activity was much earlier than the structural
loss of the molecule during the course of unfolding, with
the midpoint concentrations being 4.5 and 7.0 M for activity and spectroscopic measurements, respectively.
The equilibrium unfolding transition could be adequately
fitted to a three-state, two-step model (N 7 I 7
U) and the intermediate fraction was maximally populated
at 6.3 M urea. The free energy change (DGH2O) for
the unfolding of native (N) to intermediate (I) was 14.2 6
0.28 kcal/mol and for the intermediate to the unfolded
state (U) was 11.9 6 0.12 kcal/mol. The ANS binding
measurements as a function of urea concentration indicated
that the maximum binding of ANS was in 6.3 M
urea due to the exposure of hydrophobic groups; this
intermediate showed significant amount of tertiary
structure and retained nearly 60% of secondary structure.
The limited proteolysis measurements showed that
the initiation of unfolding was from the C-terminal domain.
Thus, the stable intermediate observed could be
the C-terminal domain unfolded with exposed hydrophobic
domain-domain interface. Limited proteolysis
experiments during refolding process suggested that
the intermediate refolded prior to completely unfolded
LOX1. These results confirmed the role of cysteine residues
and domain-domain interactions in the reversible
unfolding of LOX1. This is the first report of the reversible unfolding of a very large monomeric, multi-domain protein, which also has a prosthetic group.
 
Date 1999-12
 
Type Article
PeerReviewed
 
Format application/pdf
 
Language en
 
Identifier http://ir.cftri.com/1616/1/J._Biol._Chem.%2C_Dec_1999_274_35351_-_35358.pdf
Sudharshan, E and Appu Rao, A. G. (1999) Involvement of Cysteine Residues and Domain Interactions in the Reversible Unfolding of Lipoxygenase-1. Journal of Biological Chemistry, 274 (50). pp. 35351-35358.