The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.
A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.
van Mierlo
C P
CP
Department of Biomolecular Sciences, Laboratory of Biochemistry, Wageningen Agricultural University, The Netherlands. carlo.vanmierlo@nmr.bc.wau.nl
van Dongen
W M
WM
Vergeldt
F
F
van Berkel
W J
WJ
Steensma
E
E
D001059:Apoproteins / Q000737:chemistry*; D016948:Azotobacter vinelandii / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004355:Drug Stability; D005418:Flavodoxin / Q000737:chemistry*; D019791:Guanidine; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001059:Apoproteins; D005418:Flavodoxin; C026932:apoflavodoxin; D019791:Guanidine