Aggregation and fibrillization of the recombinant human prion protein huPrP90-231

According to the "protein-only" hypothesis, the critical step in the pathogenesis of prion diseases is the conformational transition between the normal (PrP(C)) and pathological (PrP(Sc)) isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90-231 of the human prion protein (huPrP90-231). Incubation of the protein under acidic conditions (pH 3.6-5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an alpha-helical conformation to a beta-sheet structure and oligomerization of huPrP90-231 into large molecular weight aggregates. No stable monomeric beta-sheet-rich folding intermediate of the protein could be detected in the present experiments. Kinetic analysis of the data indicates that the formation of beta-sheet structure and protein oligomerization likely occur concomitantly. The beta-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrP(Sc)). Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrP(Sc)-like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90-231 to an oligomeric beta-sheet structure is the presence of salt.     

Membrane environment alters the conformational structure of the recombinant human prion protein

The prion protein (PrP) in a living cell is associated with cellular membranes. However, all previous biophysical studies with the recombinant prion protein have been performed in an aqueous solution. To determine the effect of a membrane environment on the conformational structure of PrP, we studied the interaction of the recombinant human prion protein with model lipid membranes. The protein was found to bind to acidic lipid-containing membrane vesicles. This interaction is pH-dependent and becomes particularly strong under acidic conditions. Spectroscopic data show that membrane binding of PrP results in a significant ordering of the N-terminal part of the molecule. The folded C-terminal domain, on the other hand, becomes destabilized upon binding to the membrane surface, especially at low pH. Overall, these results show that the conformational structure and stability of the recombinant human PrP in a membrane environment are substantially different from those of the free protein in solution. These observations have important implications for understanding the mechanism of the conversion between the normal (PrP(C)) and pathogenic (PrP(Sc)) forms of prion protein.     

pH-dependent prion protein conformation in classical Creutzfeldt-Jakob disease.

In transmissible spongiform encephalopathies, the cellular prion protein (PrP(C)) undergoes a conformational change from a prevailing alpha-helical structure to a beta-sheet-rich, protease-resistant isoform, termed PrP(Sc). PrP(C) has two characteristics: a high affinity for Cu(2+) and a strong pH-dependent conformation. Lines of evidence indicate that PrP(Sc) conformation is dependent on copper and that acidic conditions facilitate the conversion of PrP(C) --> PrP(Sc). In each species, PrP(Sc) exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrP(Sc) have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrP(Sc) under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrP(Sc), associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrP(Sc) types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrP(Sc) in sCJD.     

Checking the pH-induced conformational transition of prion protein by molecular dynamics simulations: effect of protonation of histidine residues

The role of acidic pH in the conversion of human prion protein to the pathogenic isoform is investigated by means of molecular dynamics simulations, focusing the attention on the effect of protonation of histidine residues on the conformational behavior of human PrPC globular domain. Our simulations reveal a significant loss of alpha-helix content under mildly acidic conditions, due to destructuration of the C-terminal part of HB (thus suggesting a possible involvement of HB into the conformational transition leading to the pathogenic isoform) and a transient lengthening of the native beta-sheet. Protonation of His-187 and His-155 seems to be crucial for the onset of the conformational rearrangement. This finding can be related to the existence of a pathogenic mutation, H187R, which is associated with GSS syndrome. Finally, the relevance of our results for the location of a Cu2+-binding pocket in the C-terminal part of the prion is discussed.     

Assembly of the full-length recombinant mouse prion protein I. Formation of soluble oligomers

The conversion of a monomeric alpha-helix-rich isoform to multimeric beta-sheet-rich isoforms is a prominent feature of the conversion between PrP(C) and PrP(SC). We mimicked this process in vitro by exposing an unglycosylated recombinant form of the full-length mouse prion protein ((Mo)PrP(23-231)) to an acidic pH, at 37 degrees C, and we monitored the kinetics of conformational change and assembly. In these conditions, monomeric (Mo)PrP(23-231) converts slowly to two ensembles of soluble oligomers that are separated by size exclusion chromatography. The larger oligomers (I) are unstable, and their formation involves almost no change in secondary structure content. The smaller oligomers (II) form stable spherical or annular particles containing between 8 and 15 monomers as determined by multi-angle laser light scattering (MALLS). Their formation is concomitant with the main, thought limited, change in the secondary structure content (10%) seen by Fourier Transform Infrared (FTIR) spectroscopy. Even if these oligomers conserve a large part of the secondary structure of monomeric PrP, they exhibit amyloid features with the appearance of intermolecular beta-structure as revealed by the appearance of an IR band below 1620 cm(-1).     

Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red

The fluorescence of Nile red (9-diethylamino-5H-benzophenoxazine-5-one) is quenched in aqueous solutions but shows augmented fluorescence in hydrophobic environments. Nile red fluorescence was blue shifted and strongly augmented in the presence of various amyloid fibrils assayed under acidic as well as neutral pH conditions. Fibrils grown from lysozyme and insulin (at pH 1.6 and 65 °C), transthyretin (TTR) fibrils grown from the acid unfolded monomer (pH 2.0, 21 °C) or from the dissociated tetramer starting from native protein under less acidic conditions (pH 4.4, 37 °C) were detected. Nile red was also successfully employed in detecting Aβ1-42 and human prion protein (PrP90-231) amyloid fibrils grown at neutral pH. Nile red was amyloid fibril specific and did not fluoresce appreciably in the presence of the monomeric precursor proteins. Stoke's shifts of the wavelength maximum of Nile red bound to various fibrils were different (ranging from 615 nm to 638 nm) indicating sensitivity to the tertiary structure in its respective binding sites of different amyloid proteins. A polarity assay using ethanol-water mixtures and pure octanol ranging from dielectric constants between 10 and 70 showed a linear correlation of Nile red Stoke's shift and allowed assignment of amyloid fibril binding site polarity. Fluorescence resonance energy transfer between Thioflavin T (ThT) and Nile red was proven to be efficient and co-staining was employed to discriminate between conformational isoforms of Aβ1-42 amyloid fibrils grown under agitated and quiescent conditions. This paper demonstrates the complementary use of this fluorometric method for conformational typing of amyloid structures.     

On the mechanism of alpha-helix to beta-sheet transition in the recombinant prion protein

It is believed that the critical event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of the prion protein from an alpha-helical form, PrP(C), to a beta-sheet-rich conformer, PrP(Sc). Recently, we have shown that incubation of the recombinant prion protein under mildly acidic conditions (pH 5 or below) in the presence of low concentrations of guanidine hydrochloride results in a transition to PrP(Sc)-like beta-sheet-rich oligomers that show fibrillar morphology and an increased resistance to proteinase K digestion [Swietnicki, W., Morillas, M, Chen, S., Gambetti, P., and Surewicz, W. K. (2000) Biochemistry 39, 424-431]. To gain insight into the mechanism of this transition, in the present study we have characterized the biophysical properties of the recombinant human prion protein (huPrP) at acidic pH in the presence of urea and salt. Urea alone induces unfolding of the protein but does not result in protein self-association or a conversion to beta-sheet structure. However, a time-dependent transition to beta-sheet structure occurs upon addition of both urea and NaCl to huPrP, even at a sodium chloride concentration as low as 50 mM. This transition occurs concomitantly with oligomerization of the protein. At a given protein and sodium chloride concentration, the rate of monomeric alpha-helix to oligomeric beta-sheet transition is strongly dependent on the concentration of urea. Low and medium concentrations of the denaturant accelerate the reaction, whereas strongly unfolding conditions are not conducive to the conversion of huPrP into an oligomeric beta-sheet-rich structure. The present data strongly suggest that partially unfolded intermediates may be involved in the transition of the monomeric recombinant prion protein into the oligomeric scrapie-like form.     

Folding intermediates of the prion protein stabilized by hydrostatic pressure and low temperature

Prion diseases are associated with conformational conversion of the cellular prion protein, PrPC, into a misfolded form, PrPSc. We have investigated the equilibrium unfolding of the structured domain of recombinant murine prion protein, comprising residues 121-231 (mPrP-(121-231)). The equilibrium unfolding of mPrP-(121-231) by urea monitored by intrinsic fluorescence and circular dichroism (CD) spectroscopies indicated a two-state transition, without detectable folding intermediates. The fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid (bis-ANS) binds to native mPrP-(121-231), indicating exposure of hydrophobic domains on the protein surface. Increasing concentrations of urea (up to 4 M) caused the release of bound bis-ANS, whereas changes in intrinsic fluorescence and CD of mPrP took place only above 4 M urea. This indicates the existence of a partially unfolded conformation of mPrP, characterized by loss of bis-ANS binding and preservation of the overall structure of the protein, stabilized at low concentrations of urea. Hydrostatic pressure and low temperatures were also used to stabilize partially folded intermediates that are not detectable in the presence of chemical denaturants. Compression of mPrP to 3.5 kbar at 25 degrees C and pH 7 caused a slight decrease in intrinsic fluorescence emission and an 8-fold increase in bis-ANS fluorescence. Lowering the temperature to -9 degrees C under pressure reversed the decrease in intrinsic fluorescence and caused a marked (approximately 40-fold) increase in bis-ANS fluorescence. The increase in bis-ANS fluorescence at low temperatures was similar to that observed for mPrP at 1 atm at pH 4. These results suggest that pressure-assisted cold denaturation of mPrP stabilizes a partially folded intermediate that is qualitatively similar to the state obtained at acidic pH. Compression of mPrP in the presence of a subdenaturing concentration of urea stabilized another partially folded intermediate, and cold denaturation under these conditions led to complete unfolding of the protein. Possible implications of the existence of such partially folded intermediates in the folding of the prion protein and in the conversion to the PrPSc conformer are discussed.     

Preventing misfolding of the prion protein by trimethylamine N-oxide

Transmissible spongiform encephalopathies are a class of fatal neurodegenerative diseases linked to the prion protein. The prion protein normally exists in a soluble, globular state (PrP(C)) that appears to participate in copper metabolism in the central nervous system and/or signal transduction. Infection or disease occurs when an alternatively folded form of the prion protein (PrP(Sc)) converts soluble and predominantly alpha-helical PrP(C) into aggregates rich in beta-structure. The structurally disordered N-terminus adopts beta-structure upon conversion to PrP(Sc) at low pH. Chemical chaperones, such as trimethylamine N-oxide (TMAO), can prevent formation of PrP(Sc) in scrapie-infected mouse neuroblastoma cells [Tatzelt, J., et al. (1996) EMBO J. 15, 6363-6373]. To explore the mechanism of TMAO protection of PrP(C) at the atomic level, molecular dynamics simulations were performed under conditions normally leading to conversion (low pH) with and without 1 M TMAO. In PrP(C) simulations at low pH, the helix content drops and the N-terminus is brought into the small native beta-sheet, yielding a PrP(Sc)-like state. Addition of 1 M TMAO leads to a decreased radius of gyration, a greater number of protein-protein hydrogen bonds, and a greater number of tertiary contacts due to the N-terminus forming an Omega-loop and packing against the structured core of the protein, not due to an increase in the level of extended structure as with the PrP(C) to PrP(Sc) simulation. In simulations beginning with the "PrP(Sc)-like" structure (derived from PrP(C) simulated at low pH in pure water) in 1 M TMAO, similar structural reorganization at the N-terminus occurred, disrupting the extended sheet. The mechanism of protection by TMAO appears to be exclusionary in nature, consistent with previous theoretical and experimental studies. The TMAO-induced N-terminal conformational change prevents residues that are important in the conversion of PrP(C) to PrP(Sc) from assuming extended sheet structure at low pH.     

Isolation of isoforms of mouse prion protein with PrP(SC)-like structural properties.

Three novel conformational isomers of mouse prion protein mPrP(23-231) were prepared by incubating the reduced mPrP(23-231) in the presence of urea at mild acidic conditions. They are stable isomers that can be separated and isolated by reversed phase HPLC. These isomers, designated mPrP-a, mPrP-b, and mPrP-c, all exist in reduced state and monomeric form. They all exhibit a high content of beta-sheet structure upon oligomerization at near-neutral pH. They are also partially resistant to proteolysis by proteinase K and chymotrypsin. These structural properties are hallmarks of pathogenic prion protein (PrP(SC)).     

Influence of pH on NMR structure and stability of the human prion protein globular domain

The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121-230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three alpha-helices, comprising residues 144-156, 174-194, and 200-228, and a short anti-parallel beta-sheet, comprising residues 128-131 and 161-164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix alpha1 is elongated at the C-terminal end with residues 153-156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix alpha 2 is increased. The protonation of His155 and His187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121-230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154-157 at the C-terminal end of helix alpha 1 and residues 161-164 of beta-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a "starting point" for pH-induced unfolding and thus may be implicated in endosomic PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopathies.     

Sheep prion protein synthetic peptide spanning helix 1 and beta-strand 2 (residues 142-166) shows beta-hairpin structure in solution

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.     

Slow spontaneous ��-to-�� structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Acid-induced Molten Globule State of a Prion Protein: CRUCIAL ROLE OF STRAND 1-HELIX 1-STRAND 2 SEGMENT*

The conversion of a cellular prion protein (PrP^C) to its pathogenic isoform (PrP^Sc) is a critical event in the pathogenesis of prion diseases. Pathogenic conversion is usually associated with the oligomerization process; therefore, the conformational characteristics of the pre-oligomer state may provide insights into the conversion process. Previous studies indicate that PrP^C is prone to oligomer formation at low pH, but the conformation of the pre-oligomer state remains unknown. In this study, we systematically analyzed the acid-induced conformational changes of PrP^C and discovered a unique acid-induced molten globule state at pH 2.0 termed the “A-state.” We characterized the structure of the A-state using far/near-UV CD, 1-anilino-8-naphthalene sulfonate fluorescence, size exclusion chromatography, and NMR. Deuterium exchange experiments with NMR detection revealed its first unique structure ever reported thus far; i.e. the Strand 1-Helix 1-Strand 2 segment at the N terminus was preferentially unfolded, whereas the Helix 2-Helix 3 segment at the C terminus remained marginally stable. This conformational change could be triggered by the protonation of Asp¹⁴⁴, Asp¹⁴⁷, and Glu¹⁹⁶, followed by disruption of key salt bridges in PrP^C. Moreover, the initial population of the A-state at low pH (pH 2.0–5.0) was well correlated with the rate of the β-rich oligomer formation, suggesting that the A-state is the pre-oligomer state. Thus, the specific conformation of the A-state would provide crucial insights into the mechanisms of oligomerization and further pathogenic conversion as well as facilitating the design of novel medical chaperones for treating prion diseases.     

Prion protein amyloid formation under native-like conditions involves refolding of the C-terminal alpha-helical domain

Transmissible spongiform encephalopathies are associated with conformational conversion of the cellular prion protein, PrP(C), into a proteinase K-resistant, amyloid-like aggregate, PrP(Sc). Although the structure of PrP(Sc) remains enigmatic, recent studies have afforded increasingly detailed characterization of recombinant PrP amyloid. However, all previous studies were performed using amyloid fibrils formed in the presence of denaturing agents that significantly alter the folding state(s) of the precursor monomer. Here we report that PrP amyloid can also be generated under physiologically relevant conditions, where the monomeric protein is natively folded. Remarkably, site-directed spin labeling studies reveal that these fibrils possess a beta-core structure nearly indistinguishable from that of amyloid grown under denaturing conditions, where the C-terminal alpha-helical domain of the PrP monomer undergoes major refolding to a parallel and in-register beta-structure upon conversion. The structural similarity of fibrils formed under drastically different conditions strongly suggests that the common beta-sheet architecture within the approximately 160-220 core region represents a distinct global minimum in the PrP conversion free energy landscape. We also show that the N-terminal region of fibrillar PrP displays conformational plasticity, undergoing a reversible structural transition with an apparent pK(a) of approximately 5.3. The C-terminal region, on the other hand, retains its beta-structure over the pH range 1-11, whereas more alkaline buffer conditions denature the fibrils into constituent PrP monomers. This profile of pH-dependent stability is reminiscent of the behavior of brain-derived PrP(Sc), suggesting a substantial degree of structural similarity within the beta-core region of these PrP aggregates.     

The state of the prion

There is little doubt that the main component of the transmissible agent of spongiform encephalopathies - the prion - is a conformational variant of the ubiquitous host protein PrP(C), and that the differing properties of various prion strains are associated with different abnormal conformations of this protein. The precise structure of the prion is not yet known, nor are the mechanisms of infection, conformational conversion and pathogenesis understood.     

Slow spontaneous α-to-β structural conversion in a non-denaturing neutral condition reveals the intrinsically disordered property of the disulfide-reduced recombinant mouse prion protein

In prion diseases, the normal prion protein is transformed by an unknown mechanism from a mainly α-helical structure to a β-sheet-rich, disease-related isomer. In this study, we surprisingly found that a slow, spontaneous α-to-coil-to-β transition could be monitored by circular dichroism spectroscopy in one full-length mouse recombinant prion mutant protein, denoted S132C/N181C, in which the endogenous cysteines C179 and C214 were replaced by Ala and S132 and N181 were replaced by Cys, during incubation in a non-denaturing neutral buffer. No denaturant was required to destabilize the native state for the conversion. The product after this structural conversion is toxic β-oligomers with high fluorescence intensity when binding with thioflavin T. Site-directed spin-labeling ESR data suggested that the structural conversion involves the unfolding of helix 2. After examining more protein mutants, it was found that the spontaneous structural conversion is due to the disulfide-deletion (C to A mutations). The recombinant wild-type mouse prion protein could also be transformed into β-oligomers and amyloid fibrils simply by dissolving and incubating the protein in 0.5 mM NaOAc (pH 7) and 1 mM DTT at 25°C with no need of adding any denaturant to destabilize the prion protein. Our findings indicate the important role of disulfide bond reduction on the structural conversion of the recombinant prion protein, and highlight the special “intrinsically disordered” conformational character of the recombinant prion protein.     

Possible role of region 152-156 in the structural duality of a peptide fragment from sheep prion protein

The conformational conversion of the nonpathogenic "cellular" prion isoform into a pathogenic "scrapie" protease-resistant isoform is a fundamental event in the onset of transmissible spongiform encephalopathies (TSE). During this pathogenic conversion, helix H1 and its two flanking loops of the normal prion protein are thought to undergo a conformational transition into a beta-like structure. A peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. This peptide in aqueous solution, in contrast to many prion fragments studied earlier (1) is highly soluble and (2) does not aggregate until the millimolar concentration range, and (3) exhibits an intrinsic propensity to a beta-hairpin-like conformation at neutral pH. We found that this peptide can also fold into a helix H1 conformation when dissolved in a TFE/PB mixture. The structures of the peptide calculated by MD showed solvent-dependent internal stabilizing forces of the structures and evidenced a higher mobility of the residues following the end of helix H1. These data suggest that the molecular rearrangement of this peptide in region 152-156, particularly in position 155, could be associated with the pathogenic conversion of the prion protein.     

Molecular dynamics simulation of dimeric and monomeric forms of human prion protein: insight into dynamics and properties

A central theme in prion protein research is the detection of the process that underlies the conformational transition from the normal cellular prion form (PrP(C)) to its pathogenic isoform (PrP(Sc)). Although the three-dimensional structures of monomeric and dimeric human prion protein (HuPrP) have been revealed by NMR spectroscopy and x-ray crystallography, the process underlying the conformational change from PrP(C) to PrP(Sc) and the dynamics and functions of PrP(C) remain unknown. The dimeric form is thought to play an important role in the conformational transition. In this study, we performed molecular dynamics (MD) simulations on monomeric and dimeric HuPrP at 300 K and 500 K for 10 ns to investigate the differences in the properties of the monomer and the dimer from the perspective of dynamic and structural behaviors. Simulations were also undertaken with Asp178Asn and acidic pH, which is known as a disease-associated factor. Our results indicate that the dynamics of the dimer and monomer were similar (e.g., denaturation of helices and elongation of the beta-sheet). However, additional secondary structure elements formed in the dimer might result in showing the differences in dynamics and properties between the monomer and dimer (e.g., the greater retention of dimeric than monomeric tertiary structure).     

Conformational pH dependence of intermediate states during oligomerization of the human prion protein

Intermediate states are key to understanding the molecular mechanisms governing protein misfolding. The human prion protein (PrP) can follow various misfolding pathways, and forms a soluble beta-sheet-rich oligomer under acidic, mildly denaturing, high salt conditions. Here we describe a fast conformational switch from the native alpha-monomer to monomeric intermediate states under oligomer-forming conditions, followed by a slower oligomerization process. We observe a pH dependence of the secondary structure of these intermediate forms, with almost native-like alpha-helical secondary structure at pH 4.1 and predominantly beta-sheet characteristics at pH 3.6. NMR spectroscopy differentiates these intermediate states from the native protein and indicates dynamic rearrangements of secondary structure elements characteristic of a molten globule. The alpha-helical intermediate formed at pH 4.1 can convert to the beta-sheet conformation at pH 3.6 but not vice versa, and neither state can be reconverted to an alpha-monomer. The presence of methionine rather than valine at codon 129 accelerates the rate of oligomer formation from the intermediate state.