Volume 272, Number 11,
Issue of March 14, 1997
pp. 7099-7105
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Equilibrium Intermediates in the Reversible Unfolding of Firefly
(Photinus pyralis) Luciferase
(Received for publication, July 31, 1996, and in revised form, January 6, 1997)
Ruth
Herbst
,
Ute
Schäfer
and
Robert
Seckler
From the Universität Regensburg, Institut für Biophysik
und Physikalische Biochemie, D-93040 Regensburg, Germany
Firefly luciferase has been used as a model
protein to study cotranslational and chaperone-assisted protein
folding. We found conditions for reversible unfolding of luciferase in
the absence of cellular factors, and we characterized
denaturant-induced equilibrium unfolding transitions and refolding
kinetics of the enzyme. Luciferase unfolding induced by guanidinium
chloride at 10 °C can be described as a four-state equilibrium with
two inactive intermediates highly populated around 1 and 3 M denaturant. The transitions occur around 0.3, 1.7, and
3.8 M denaturant. The free energy of denaturation to the
first inactive intermediate
(
G0N
I1 = 15 ± 3 kJ·mol
1) is small for a protein of 60 kDa. Fluorescence
and circular dichroism spectra of the intermediates indicate that
I1 has a compact conformation, whereas aromatic side chains
are highly exposed in the second intermediate, I2, despite
its high content of secondary structure. In the presence of a
hydrophilic detergent, significant reactivation of luciferase is
observed up to temperatures at which the native protein is unstable.
Reactivation kinetics of luciferase are exceedingly slow and probably
not limited by proline isomerization, as suggested by their
independence from the time spent in the unfolded state.
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