Thermodynamic Analysis of Denaturant-Induced Unfolding of HodC69S Protein Supports a Three-State Mechanism

Thermodynamic stability parameters and the equilibrium unfolding mechanism of His6HodC69S, a mutant of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) having a Cys to Ser exchange at position 69 and an N-terminal hexahistidine tag (His6HodC69S), have been derived from isothermal unfolding studies...

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Published in:Biochemistry (Easton) 2008-07, Vol.47 (27), p.7116-7126
Main Authors: Boehm, Kristian, Guddorf, Jessica, Albers, Alexander, Kamiyama, Tadashi, Fetzner, Susanne, Hinz, H.-J
Format: Article
Language:English
Subjects:
Arthrobacter - drug effects
Arthrobacter - enzymology
Arthrobacter - radiation effects
Circular Dichroism
Dioxygenases - chemistry
Dioxygenases - metabolism
Disulfides - chemistry
Light
Mutant Proteins - chemistry
Mutant Proteins - metabolism
Oxidation-Reduction - drug effects
Oxidation-Reduction - radiation effects
Protein Denaturation - drug effects
Protein Denaturation - radiation effects
Protein Folding
Scattering, Radiation
Spectrometry, Fluorescence
Thermodynamics
Urea - pharmacology
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Summary:Thermodynamic stability parameters and the equilibrium unfolding mechanism of His6HodC69S, a mutant of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) having a Cys to Ser exchange at position 69 and an N-terminal hexahistidine tag (His6HodC69S), have been derived from isothermal unfolding studies using guanidine hydrochloride (GdnHCl) or urea as denaturants. The conformational changes were monitored by following changes in circular dichroism (CD), fluorescence, and dynamic light scattering (DLS), and the resulting transition curves were analyzed on the basis of a sequential three-state model N = I = D. The structural changes have been correlated to catalytic activity, and the contribution to stability of the disulfide bond between residues C37 and C184 in the native protein has been established. A prominent result of the present study is the finding that, independent of the method used for denaturing the protein, the unfolding mechanism always comprises three states which can be characterized by, within error limits, identical sets of thermodynamic parameters. Apparent deviations from three-state unfolding can be rationalized by the inability of a spectroscopic probe to discriminate clearly between native, intermediate, and unfolded ensembles. This was the case for the CD-monitored urea unfolding curve.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi800554v