Abstract
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 degrees 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 (DeltaG0N <==> 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.
Highlights
The observation that some organisms are able to convert chemical energy into visible light has been fascinating researchers for many years
We found that firefly luciferase does refold spontaneously and with high yield at low temperature and low protein concentration, conditions chosen to minimize aggregation as a competing side reaction
This allowed us to determine the thermodynamic stability of the enzyme from equilibrium unfolding transitions, to physically characterize two highly populated folding intermediates, and to follow the kinetics of luciferase refolding
Summary
The observation that some organisms are able to convert chemical energy into visible light has been fascinating researchers for many years. It is generally held that the information required for correct folding is entirely encoded in the amino acid sequence of a protein [16], and proteins found to depend on molecular chaperones for efficient folding in vivo generally can be efficiently renatured if refolding conditions are carefully optimized [17] Such refolding experiments are a prerequisite for understanding critical steps in protein-folding pathways and to identify folding intermediates as candidate targets of chaperone action. We found that firefly luciferase does refold spontaneously and with high yield at low temperature and low protein concentration, conditions chosen to minimize aggregation as a competing side reaction This allowed us to determine the thermodynamic stability of the enzyme from equilibrium unfolding transitions, to physically characterize two highly populated folding intermediates, and to follow the kinetics of luciferase refolding
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