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The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate
Author(s) -
Van Mierlo Carlo P. M.,
Van Dongen Walter M. A. M.,
Vergeldt Frank,
Van Berkel Willem J. H.,
Steensma Elles
Publication year - 1998
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560071110
Subject(s) - azotobacter vinelandii , flavodoxin , circular dichroism , chemistry , molten globule , crystallography , protein folding , biochemistry , ferredoxin , organic chemistry , nitrogenase , nitrogen fixation , nitrogen , enzyme
A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of α/ß 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.