In this study, the heat induced fibrilar aggregation of the whey protein beta-lactoglobulin is investigated at low pH and at low ionic strength. Under these circumstances, tapping mode atomic force microscopy results indicate that the fibrils formed have a periodic structure with a period of about 25 nm and a thickness of one or two protein monomers. Fibril formation is followed in situ using light scattering and proton NMR techniques. The dynamic light scattering results show that the fibrils that form after short heating periods (up to a few hours) disintegrate upon slow cooling, whereas fibrils that form during long heating periods do not disintegrate upon subsequent slow cooling. The NMR results show that even after prolonged heating an appreciable fraction of the protein molecules is incorporated into fibrils only when the beta-lactoglobulin concentration is above approximately 2.5 wt %. The data imply multiple steps during the heat induced formation of beta-lactoglobulin fibrils at low pH and at low ionic strength: (partly) denatured protein monomers are either incorporated into fibrils or form instead a low molecular weight complex that is incapable of forming fibrils. Fibril formation itself also involves (at least) two steps: the reversible formation of linear aggregates, followed by a slow process of "consolidation" after which the fibrils no longer disintegrate upon slow cooling.

The folding kinetics of the 179-residue Azotobacter vinelandii apoflavodoxin, which has an alpha-beta parallel topology, have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy. Single-jump and interrupted refolding experiments show that the refolding kinetics involve four processes yielding native molecules. Interrupted unfolding experiments show that the two slowest folding processes are due to Xaa-Pro peptide bond isomerization in unfolded apoflavodoxin. The denaturant dependence of the folding kinetics is complex. Under strongly unfolding conditions (>2.5 M GuHCl), single exponential kinetics are observed. The slope of the chevron plot changes between 3 and 5 M denaturant, and no additional unfolding process is observed. This reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate. Under refolding conditions, two processes are observed for the folding of apoflavodoxin molecules with native Xaa-Pro peptide bond conformations, which implies the population of an intermediate. The slowest of these two processes becomes faster with increasing denaturant concentration, meaning that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. It is shown that the intermediate that populates during refolding is off-pathway. The experimental data obtained on apoflavodoxin folding are consistent with the linear folding mechanism I(off) <==> U <==> I(on) <== > N, the off-pathway intermediate being the molten globule one that also populates during equilibrium denaturation of apoflavodoxin. The presence of such on-pathway and off-pathway intermediates in the folding kinetics of alpha-beta parallel proteins is apparently governed by protein topology.

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...

Detailed information about unfolded states is required to understand how proteins fold. Knowledge about folding intermediates formed subsequently is essential to get a grip on pathological aggregation phenomena. During folding of apoflavodoxin, which adopts the widely prevalent alpha-beta parallel topology, most molecules fold via an off-pathway folding intermediate with helical properties. To better understand why this species is formed, guanidine hydrochloride-unfolded apoflavodoxin is characterized at the residue level using heteronuclear NMR spectroscopy. In 6.0 M denaturant, the protein behaves as a random coil. In contrast, at 3.4 M denaturant, secondary shifts and (1)H-(15)N relaxation rates report four transiently ordered regions in unfolded apoflavodoxin. These regions have restricted flexibility on the (sub)nanosecond time scale. Secondary shifts show that three of these regions form alpha-helices, which are populated about 10% of the time, as confirmed by far-UV CD data. One region of unfolded apoflavodoxin adopts non-native structure. Of the alpha-helices observed, two are present in native apoflavodoxin as well. A substantial part of the third helix becomes beta-strand while forming native protein. Chemical shift changes due to amino acid residue replacement show that the latter alpha-helix has hydrophobic interactions with all other ordered regions in unfolded apoflavodoxin. Remarkably, these ordered segments dock non-natively, which causes strong competition with on-pathway folding. Thus, rather than directing productive folding, conformational preorganization in the unfolded state of an alpha-beta parallel-type protein promotes off-pathway species formation.

Although adsorption-induced conformational changes of proteins play an essential role during protein adsorption on interfaces, detailed information about these changes is lacking. To further the current understanding of protein adsorption, in this study, the orientation, conformation, and local stability of bovine ␣-lactalbumin (BLA) adsorbed on polystyrene nanospheres is characterized at the residue level by hydrogen͞deuterium exchange and 2D NMR spectroscopy. Most of the adsorbed BLA molecules have conformational properties similar to BLA molecules in the acid-induced molten globule state (A state). A folding intermediate of BLA is thus induced and trapped by adsorption of the protein on the hydrophobic interface. Several residues, clustered on one side of the adsorbed folding intermediate of BLA, have altered amide proton exchange protection factors compared to those of the A state of BLA. This side preferentially interacts with the interface and includes residues in helix C, the calcium binding site, and part of the ␤-domain. Local unfolding of this interacting part of the adsorbed protein seems to initiate the adsorption-induced unfolding of BLA. Adsorption-induced protein unfolding apparently resembles more the mechanical unfolding of a protein than the global unfolding of a protein as induced by denaturant, pH, or pressure. 2D macromolecular crowding prevented the minority of adsorbed BLA molecules, which arrived late at the interface, to unfold to the A state. Protein adsorption is a novel and challenging approach to probe features of the free energy landscapes accessible to unfolding proteins.bovine ␣-lactalbumin ͉ hydrogen͞deuterium exchange ͉ NMR A dsorption of protein molecules on solid interfaces is important in fields like biomedical materials engineering (1), chromatography (2), and nanotechnology (3, 4). To understand protein adsorption, knowledge of adsorption-induced conformational changes of proteins is essential (5, 6). Unfortunately, because of considerable experimental difficulties, detailed information at the submolecular level about these conformational changes is sparse (7-12), which hampers the further development of the theory of protein adsorption. To stimulate this development, we characterize here at the residue level, the orientation, conformation, and local stability of bovine ␣-lactalbumin (BLA) adsorbed on polystyrene nanospheres by hydrogen͞deuterium (H͞D) exchange and 2D NMR spectroscopy. BLA, a 14-kDa protein from milk, is chosen to study protein adsorption because its structure, stability, and folding behavior have been thoroughly investigated. In addition, the adsorption of BLA on a variety of interfaces has been studied.Information about the stability and dynamics of a protein at the level of single amino acids can be obtained by investigating its H͞D exchange characteristics (13). In this study, BLA molecules in the native state are added to solid polystyrene nanospheres in 2 H 2 O. All added protein molecules adsorb spontaneously and rapidly [i.e., within 15 ms (14)] on ...