A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of a/P 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.Keywords: apoflavodoxin; circular dichroism; equilibrium unfolding; fluorescence; molten globule-like species; NMR; protein stability; three-state model In contrast to most protein folds, the flavodoxin-like fold is shared by many (i.e., nine) superfamilies (Brenner, 1997 a broad range of unrelated proteins with different functions like catalases, chemotactic proteins, lipases, esterases, and flavodoxins. They are all characterized by a five-stranded parallel P-sheet surrounded by a-helices at either side of the sheet. Of these proteins, a flavodoxin is chosen by us as a model system to study protein folding and stability. General rules governing protein folding are only beginning to emerge and there is a strong need for additional well-characterized protein systems to obtain a better understanding of the fundamental rules describing protein folding. By studying the folding of flavodoxin, we extend the number of proteins under investigation and expect to learn the rules by which other, if not all, proteins with a flavodoxin-like topology fold.Flavodoxins are a group of small flavoproteins that function as low-potential one-electron carriers and contain a noncovalently bound FMN cofactor (Mayhew & Tollin, 1992). The protein investigated by us is flavodoxin 11 from Azotobacter vinelandii (strain ATCC 478), henceforth designated flavodoxin. Its gene has been cloned in Escherichia coli and brought to high expression in our laboratory. The protein consists of 179 amino acid residues and belongs to the class of "long-chain" flavodoxins (Ta...
A new method is described for direct monitoring of the conformational stability of proteins that are physically adsorbed from solution onto a solid substrate. The adsorption-induced conformational changes of insulin are studied using a combination of hydrogen/deuterium (H/D) exchange and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The effect of the surface hydrophobicity on the adsorption-induced conformational changes in the insulin structure is probed by adsorbing insulin on a hydrophilic silica and a hydrophobic methylated silica surface before subjecting the insulin molecules to the isotopic exchange process. The present study describes the experimental procedure of this new application of MALDI. Results show that insulin is more highly and more irreversibly adsorbed to a hydrophobic methylated silica surface than to a hydrophilic silica surface. Hydrogen-exchange experiments clearly demonstrate that the strong interaction of insulin with the hydrophobic surface is accompanied by a strong increase in the H/D-exchange rates and thus in a reduction in the insulin native structural stability. In contrast, H/D-exchange rates of insulin are somewhat reduced upon adsorption on silica from solution.
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