Apoflavodoxin (un) folding followed at the residue level by NMR

Abstract

The denaturant-induced~un!folding of apoflavodoxin from Azotobacter vinelandii has been followed at the residue level by NMR spectroscopy. NH groups of 21 residues of the protein could be followed in a series of 1 H-15 N heteronuclear single-quantum coherence spectra recorded at increasing concentrations of guanidinium hydrochloride despite the formation of protein aggregate. These NH groups are distributed throughout the whole apoflavodoxin structure. The midpoints of unfolding determined by NMR coincide with the one obtained by fluorescence emission spectroscopy. Both techniques give rise to unfolding curves with transition zones at significantly lower denaturant concentrations than the one obtained by circular dichroism spectroscopy. The NMR~un!folding data support a mechanism for apoflavodoxin folding in which a relatively stable intermediate is involved. Native apoflavodoxin is shown to cooperatively unfold to a molten globule-like state with extremely broadened NMR resonances. This initial unfolding step is slow on the NMR chemical shift timescale. The subsequent unfolding of the molten globule is faster on the NMR chemical shift timescale and the limited appearance of 1 H-15 N HSQC cross peaks of unfolded apoflavodoxin in the denaturant range studied indicates that it is noncooperative. Keywords: apoflavodoxin; cooperative and noncooperative unfolding; equilibrium~un!folding; guanidinium hydrochloride; molten globule; NMR; protein aggregation; residue levelFlavodoxin is an interesting model to study protein folding and stability. In contrast to most protein folds, the flavodoxin-like fold is shared by many~i.e., nine! superfamilies~Brenner, 1997!. These nine superfamilies exhibit little or no sequence similarity, and comprise a broad range of unrelated proteins with different functions such as catalases, chemotactic proteins, lipases, esterases, and flavodoxins. They are all characterized by a five-stranded parallel b-sheet surrounded by a-helices at either side of the sheet. Studies on these structurally but not sequentially related proteins are pre-eminently suitable to study protein folding and stability. By studying the folding of flavodoxin, we expect to obtain a better understanding of the fundamental rules describing the folding of proteins with a flavodoxin-like 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 II from Azotobacter vinelandii~strain ATCC 478!, henceforth designated flavodoxin. The protein consists of 179 amino acid residues and belongs to the class of "longchain" flavodoxins~Tanaka et al., 1977!. Upon removal of the flavin, apoflavodoxin is generated. Despite the removal of the FMN cofactor, large parts of the tertiary structure of holo-and apoflavodoxin are strictly conserved as is reflected among others in the identity of NMR chemical shifts~Fig. 1!. Native apoflavodoxin has a stable well-ordered core, b...

Gradual Folding of an Off-Pathway Molten Globule Detected at the Single-Molecule Level

“…For example, the GuHCl-dependent (un)folding of native apoflavodoxin gives rise to a steep change in tryptophan fluorescence emission in the corresponding transition region of (un)folding (midpoint of (un)folding Cm is 1.53 ± 0.01 M) [31]. In addition, NMR spectroscopic tracking of the unfolding of 21 residues distributed throughout native apoflavodoxin reveals coinciding midpoints of (un)folding (mean Cm of 1.48 ± 0.04 M GuHCl) [42], similar to the one obtained by tryptophan fluorescence. These experiments show a highly cooperative transition that happens due to unfolding of native apoflavodoxin, which leads to the denaturantdependent population of non-native protein.…”

Flavodoxins: sequence, folding, binding, function and beyond

“…There is, nevertheless, good evidence indicating that the FMN cofactor is not required: when the wild-type Anabaena protein is overexpressed and the cells are quickly harvested (in FMN limiting conditions), or when FMNbinding defective apoflavodoxin variants are expressed, large amounts of well-folded apoprotein are recovered (our unpublished observations and [19]). The stability of apoflavodoxin has been investigated using, in addition to the Anabaena [20][21] and Azotobacter [22][23] proteins, those of several Desulfovibrio strains and species [24][25][26]. It seems clear that the equilibrium thermal unfolding is three-state, so that a partly unfolded intermediate accumulates at moderately high temperatures (Fig.…”

“…In the last 10 years, the flavodoxin folding mechanism has been outlined [15][16], the first three-dimensional structures of apoflavodoxin have been determined [34,38], the mechanism of apoflavodoxin/FMN recognition has been actively investigated [40][41][42][43][44], the stability of both the apo and holo forms are now largely characterized [20][21][22][23][24][25][26][27], the understanding of the role of the polypeptide in shaping redox potentials is much advanced [48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63] and new flavodoxin functions, beyond those of replacing ferredoxin, have been characterized. Indeed, flavodoxin is an essential protein for the survival of certain organisms, such as the pathogen Helicobacter pylori [39].…”

Interrupted Hydrogen/Deuterium Exchange Reveals the Stable Core of the Remarkably Helical Molten Globule of α-β Parallel Protein Flavodoxin

“…Spectroscopic data show that the off-pathway folding species of flavodoxin is molten globule-like; its hydrodynamic radius is closer to the native state than to the unfolded state, its three tryptophans are solvent-exposed, and it has severely broadened NMR resonances due to exchange between different conformers on the micro-to millisecond time scale (18,29,30). An off-pathway intermediate is experimentally observed for all other ␣-␤ parallel proteins of which the kinetic folding has been investigated (i.e.…”

“…We showed that at biologically relevant protein concentrations or by mimicking molecular crowding conditions present in cells, severe aggregation of the flavodoxin off-pathway species occurs (29,30). To further the understanding of the conformational properties of the flavodoxin molten globule, in this study, H/D exchange detected by NMR spectroscopy is used as a powerful technique that gives detailed information about a protein at the level of single amino acids.…”