Transition of transferrin from native to fibrillar state: An implication for amyloid-linked diseases

Human transferrin (hTF), an α/β protein, transforms from its native soluble form to proto-fibrils and amyloid fibrils at 20% TFE after prolonged incubation. This type of amyloid fibrils is observed in a number of pathological disorders. Existence of dry molten-globule state, at 5% TFE, was characterized by native-like secondary structure, Trp fluorescence and negligible ANS binding, indicating its dry interior. At 15% TFE, decrease in Trp and increase in ANS fluorescence was observed with native-like secondary structure, indicating exposure to water molecule and hence, this was referred to as Wet MG state. AFM revealed protofibrils as smaller in size howbeit amyloid fibrils were long and stiffer in morphology. Amyloid fibrils were found to possess cross-linked β-sheet, lack of tertiary contacts, as revealed by CD and ATR-FTIR, enhanced Thioflavin T fluorescence and shift in Congo red absorbance. These results showed that formation of amyloid fibrils becomes favorable when protein is destabilized in suitable conditions and non-covalent interactions, particularly intermolecular hydrogen bonding becomes prominent. Protofibrils were genotoxic in nature albeit amyloid fibrils lack this effect.     

Structural intermediates of acid unfolded Con-A in different co-solvents: fluoroalcohols and polyethylene glycols

A molten globule-like intermediate of Con-A was obtained when subjected to acid unfolding. At pH 2 the intermediate was found to have native-like secondary structure, somewhat denatured tertiary structure and maximum ANS binding. Further the stability of this intermediate was studied in presence of fluoroalcohols (TFE and HFIP) and polyethylene glycols (PEG-400, 4000 and 20,000). Secondary structural changes were monitored by far-UV CD while alterations in the tertiary structure of the acid unfolded intermediate were probed by near-UV CD. To study the environment and position of the tryptophan residues present intrinsic fluorescence studies were performed. ANS binding studies were also made to know the extent of exposure of the hydrophobic patches. Using the above-mentioned techniques it was found that in presence of fluoroalcohols the pH 2 intermediate was transformed to a state with predominant alpha-helical secondary and denatured tertiary structures. In the pathway of these transformations MG-like intermediates were formed at 10% TFE and 6% HFIP. The folding intermediate of Con-A obtained at pH 2 underwent a series of conformational changes when exposed to different molecular weight PEGs. Secondary structure was induced by low molecular weight PEG-400 and low concentrations of PEG-4000 and PEG-20,000 while at higher concentrations transition in structure was observed. Tertiary structure was stabilized only at low concentrations of PEG-400. PEG-4000 and PEG-20,000 in the whole concentration range resulted in the loss of tertiary structure.     

Cosolvents Induced Unfolding and Aggregation of Keyhole Limpet Hemocyanin

The objective of this study was to examine the effects of 2,2,2 trifluoroethanol (TFE) and acetonitrile (ACN) on the stability, behavior, and structural characteristics of giant multimeric protein Keyhole Limpet hemocyanin (KLH) by combining the circular dichroism (CD) and fluorescence measurements of KLH solution. In concentration range 20–50 % (v/v) TFE, protein at pH 7.4 shows visible aggregation while no aggregation was observed in the entire concentration range of TFE at molten globule (MG) state (pH 2.8) and resulted in stable α-helix. Our result shows that in the presence of 80 % (v/v) and 40 % (v/v) TFE, at native (pH 7.4) and MG state (pH 2.8) occurred in a highly helical state referred to as TFE denatured state I and II, respectively. However, in case of ACN, aggregation starts above 40 % (v/v) for pH 7.4 and at 80 % (v/v) for acid-induced MG (pH 2.8) state, which was dominated by β-sheet structure and referred to as ACN denatured state III and IV. An important object of our investigation is to get more detail study of efficiency of cosolvents in inducing structural changes in KLH. The dependence of scattering intensity and the R _h on alcohol concentrations was investigated at 25 °C.     

A unique molten globule state occurs during unfolding of cytochrome c by LiClO4 near physiological pH and temperature: structural and thermodynamic characterization

We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO4 at pH 6.0 and 25 degrees C by observing changes in difference molar absorbance at 400 nm (Deltaepsilon400), mean residue ellipticities at 222 nm ([theta]222) and difference mean residue ellipticity at 409 nm (Delta[theta]409). The denaturation is a three-step process when measured by Deltaepsilon400 and Delta[theta]409, and it is a two-step process when monitored by [theta]222. The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO4-induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl2) suggests that LiClO4 induces a unique MG state, i.e., (i) the core in the LiClO4-induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO4-induced process, native state <--> MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions.     

Conformational diversity of acid-denatured cytochrome c studied by a matrix analysis of far-UV CD spectra.

The singular value decomposition (SVD) analysis was applied to a large set of far-ultraviolet circular dichroism (far-UV CD) spectra (100-400 spectra) of horse heart cytochrome c (cyt c). The spectra were collected at pH 1.7-5.0 in (NH4)2SO4, sorbitol and 2,2,2-trifluoroethanol (TFE) solutions. The present purpose is to develop a rigorous matrix method applied to far-UV CD spectra to resolve in details conformational properties of proteins in the non-native (or denatured) regions. The analysis established that three basis spectral components are contained in a data set of difference spectra (referred to the spectrum of the native state) used here. By a further matrix transformation, any observed spectrum could be decomposed into fractions of the native (N), the molten-globule (MG), the highly denatured (D), and the alcohol-induced helical (H) spectral forms. This method could determine fractional transition curves of each conformer as a function of solution conditions, which gave the results consistent with denaturation curves of cyt c monitored by other spectroscopic methods. The results in sorbitol solutions, for example, suggested that the preferential hydration effect of the co-solvent stabilizes the MG conformer of cyt c. This report has found that the systematic SVD analysis of the far-UV CD spectra is a powerful tool for the conformational analysis of the non-native species of a protein when it is suitably supplemented with other experimental methods.     

Conformational Transitions Provoked by Organic Solvents in Chicken Egg Ovalbumin: Mimicking the Local Environment

Glycoprotein ovalbumin is an important protein to study helix/sheet transitions as it possess almost equal amount of α-helix and β-sheet. Conformational changes on ovalbumin at various concentrations of glyoxal, ethylene glycol (EG) and polyethylene glycol-400 (PEG-400) were investigated by fluorescence spectroscopy, circular dichroism, attenuated total reflection Fourier transform infra red spectroscopy, 8-anilino-1-naphthalenesulfonic acid and thioflavin T assay. A partially folded state of ovalbumin at 50 % v/v glyoxal was detected that preceded the onset of the aggregation process at the maximum concentration (90 % v/v) of this aldehyde. Aggregates of ovalbumin in the presence EG and PEG-400 were deduced at 70 and 80 % v/v respectively. Maximum aggregation of ovalbumin was observed at 80 % v/v PEG-400, followed by 70 % v/v EG and 90 % v/v glyoxal. Our study confirms that protein aggregation is influenced by the chemistry of organic solvent used thus follows an order of solvent effectiveness (PEG > EG > glyoxal) in inducing the transition. These results provide valuable information on the mechanisms involved in the pathogenesis of some conformational diseases. The α-helix to β-sheet conversion is a diagnostic feature of protein aggregation and has been considered as a general characteristic of amyloid fibrillogenesis in vitro.     

Molten Globule of Hemoglobin Proceeds into Aggregates and Advanced Glycated End Products

Conformational alterations of bovine hemoglobin (Hb) upon sequential addition of glyoxal over a range of 0–90% v/v were investigated. At 20% v/v glyoxal, molten globule (MG) state of Hb was observed by altered tryptophan fluorescence, high ANS binding, existence of intact heme, native-like secondary structure as depicted by far-UV circular dichroism (CD) and ATR-FTIR spectra as well as loss in tertiary structure as confirmed by near-UV CD spectra. In addition, size exclusion chromatography analysis depicted that MG state at 20% v/v glyoxal corresponded to expanded pre-dissociated dimers. Aggregates of Hb were detected at 70% v/v glyoxal. These aggregates of Hb had altered tryptophan environment, low ANS binding, exposed heme, increased β-sheet secondary structure, loss in tertiary structure, enhanced thioflavin T (ThT) fluorescence and red shifted Congo Red (CR) absorbance. On incubating Hb with 30% v/v glyoxal for 0–20 days, advanced glycation end products (AGEs) were detected on day 20. These AGEs were characterised by enhanced tryptophan fluorescence at 450 nm, exposure of heme, increase in intermolecular β-sheets, enhanced ThT fluorescence and red shift in CR absorbance. Comet assay revealed aggregates and AGEs to be genotoxic in nature. Scanning electron microscopy confirmed the amorphous structure of aggregates and branched fibrils of AGEs. The transformation of α-helix to β-sheet usually alters the normal protein to amyloidogenic resulting in a variety of protein conformational disorders such as diabetes, prion and Huntington''s.     

Transition of a Compact Intermediate State of Pea Lectin Under the Influence of Different Molecular Weight Polyethylene Glycols

The compact intermediate of the pea lectin found to exist at pH 2.4 was treated with low (PEG-400), medium (PEG-4000) and high (PEG-20,000) molecular weight PEGs. The changes occurring in the secondary structure of the protein were monitored by CD spectropolarimetry in the far-UV range, intrinsic fluorescence was used as a probe to observe the changes in the tertiary structure which is reflected by the changes in the tryptophan environment, further ANS binding studies were made to know the extent of exposure of the hydrophobic patches which is again indicative of the overall changes occurring in the tertiary structure of the protein. It was found that the three PEGs altered the secondary as well as tertiary structure of the pH 2.4 intermediate leading to the formation of three different intermediates. The intermediates were found to have non-native secondary structure as well as non-native tertiary structure. The intermediate formed by the action of PEG-400 was due to the induction of secondary and tertiary structure while the intermediates formed under the influence of PEG-4000 and PEG-20,000 were due to loss in secondary structure and rearrangement in tertiary structure. Also the ANS binding studies showed the absence of any MG or MG-like structures formed in the folding /unfolding pathway induced by PEGs.     

Trifluoroethanol and acetonitrile induced formation of the molten globule states and aggregates of cellulase

A systematic investigation on the effects of trifluoroethanol and acetonitrile at various concentrations on cellulase (EC 3.2.1.4) was studied by enzyme assay, intrinsic fluorescence, ANS binding, circular dichroism and ATR-Fourier transform infra red spectroscopy. The results show the presence of molten globule states at 3% (v/v) TFE and 80% (v/v) ACN. Cellulase aggregates at 25% (v/v) TFE and 90% (v/v) ACN, as detected by decrease in intrinsic and ANS fluorescence, loss in tertiary structure and enzyme activity, increase non-native β-sheet structure as evident from far-UV CD and FTIR spectra, increase in Thioflavin T fluorescence and shift in Congo red assay.     

Equimolar Mixture of 2,2,2-Trifluoroethanol and 4-Chloro-1-butanol is a Stronger Inducer of Molten Globule State: Isothermal Titration Calorimetric and Spectroscopic Studies

A mixture of 4-chloro-1-butanol and 2,2,2-Trifluoroethanol (TFE) has been used to generate Molten globule (MG) state of structurally homologous but functionally different proteins bovine α-lactalbumin and hen egg-white lysozyme. The thermal denaturation was done using UV–Visible spectroscopy. From UV–Visible profile, thermal transition was not observed beyond a particular concentration. There was an indication of molten globule state in case of α-lactalbumin from circular dichroism experiments. By intrinsic tryptophan fluorescence, acrylamide and potassium iodide quenching, 8-anilino-naphthalene sulfonic acid (ANS) binding and energy transfer studies the presence of molten globule state was confirmed. Quantitative characterization of MG state and determining the binding thermodynamics of ANS to the MG state was done using Isothermal Titration Calorimetry (ITC). Results show that α-lactalbumin exists in MG state at a particular concentration but lysozyme does not show features of MG state.     

Molten globule state of human placental cystatin (HPC) at low pH conditions and the effects of trifluoroethanol (TFE) and methanol.

Acid-induced conformational changes were studied in human placental cystatin (HPC) in terms of circular dichroism (CD) spectroscopy, the binding of hydrophobic dye 1-anilinonapthalene-8-sulphonic acid (ANS), and intrinsic fluorescence measurements. Our results show the formation of an acid-induced molten globule state at pH 2.0, with significant secondary and tertiary interactions that resemble the native state, exposed hydrophobic regions and the effects of trifluoroethanol (TFE) and methanol in conversion of the acid-denatured state of HPC to the alcohol-induced state, which is characterized by increased helical content, disrupted tertiary structure, and the absence of hydrophobic clusters. Alcohol-induced formation of alpha-helical structures at pH 2.0 is evident from the increase in the ellipticity values at 222 nm, with native-like secondary structural features at 40% TFE. The increase in helical content was observed up to 80% TFE concentration. The ability of TFE (40%) to refold acid-denatured HPC to native-state conformation is also supported by intrinsic and ANS fluorescence measurements.     

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T_ms), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.     

Peroxidase improves the activity of catalase by preventing aggregation during TFE-induced denaturation

Catalase, a ubiquitous enzyme of the free radical scavenging machinery unfolds and aggregates in the presence of 2,2,2-triflouroethanol (TFE). Catalase molecule aggregates at 50% TFE as evident by high thioflavin T fluorescence, shifted congo red absorbance, change in circular dichroism and soret spectra. TEM images confirmed the nature of catalase aggregates to be oligomers. Organic solvent-induced aggregation of catalase is prevented by the presence of peroxidase (another enzyme of the free radical scavenging machinery). To alter the progress of aggregation in presence of increasing concentration of TFE, we determined the effect of peroxidase on catalase oligomerization by several different techniques, including turbidity measurement, activity assay, thioflavin T fluorescence, circular dichroism, shift in congo red absorbance, transmission electron microscopy (TEM), Rayleigh scattering, soret absorption spectra, and ANS fluorescence. The presence of peroxidase in the vicinity of folded catalase helps it to remain functionally active and inhibited aggregation in the presence of TFE, suggesting that proteins are stable in crowded environments. Moreover, this catalase-peroxidase interaction is biologically significant as it provides insights into how the aggregation process may be altered.     

Spectroscopic studies on native and protofibrillar insulin

The structure of insulin in amyloid fibrillar form has been recently shown as a well folded conformation using cryoelectron microscopy [Jimenez, J.L., Nettleton, E.J., Bouchard, M., Robinson, C.V., Dobson, C.M., Saibil H.R., 2002. The protofilament structure of insulin amyloid fibrils. Proc. Natl. Acad. Sci. USA. 99 9196-9201.]. Most of the amyloid aggregates elicit maximum toxicity in the protofibrillar (PF) intermediate state. Here, we describe PF intermediates of insulin are made-up monomers with flexible conformers. We also show protofibrils have three-dimensionally extended hydrophobic cavity to bind with 1-anilinonaphthalene-8-sulphonate (ANS) molecules. Energy transfer measurement revealed that ANS dye binding site of PF is within the range of FRET distance of insulin tyrosine residues. Significant proportion of beta-sheet, helical, and turn structures in the PF form indicate conformational dynamics in the folded chain of insulin in the PF assembled form. Though the conformational flexibility is noticeably present in the assembly, addition of GdnHCl could completely unfold PF into disordered structure suggesting structural "zipping" in the PF form. We have also shown that helical conformer inducing solvent 2,2,2-trifluoroethanol (TFE) could dissociate the PF aggregate indicating possible involvement of beta-sheets in contributing to PF stability.     

Using polyethylene glycol as nonionic osmoticum to promote growth and lipid production of marine microalgae Nannochloropsis oculata

To promote the economic feasibility of Nannochloropsis oculata, efficacy of using polyethylene glycol (PEG) to increase microalgal growth and lipid accumulation was investigated. We first examined the effects of PEG concentrations on microalgal growth using 0–5 % (w/v) PEG-6000, and followed by exploring the effects of PEG molecular weights (400, 600, 2,000, 4,000, 6,000, and 20,000) on microalgal growth, size, as well as on yields of biomass, total lipids, and eicosapentaenoic acid. In addition, the capacity of PEG to reduce the effect of oxygen inhibition on microalgal growth was also investigated to evaluate its adaptability for use in large-scale and closed setting. Our results showed that PEG-induced osmotic stress (Π) in the range of 2.465–2.472 MPa can raise microalgal growth. The PEG with higher molecular weight exhibited greater efficacy of growth promotion but less lipid content under equal concentration. In this study, 0.5 % (w/v) PEG-20000 (Π = 2.466 MPa) remarkably enhanced microalgal growth without interference of intracellular lipid productivity and cellular size, yielding >50 % (w/w) increases in biomass, total lipid, and eicosapentaenoic acid amounts after 7 days that provided the optimal condition for microalgal cultivation. These positive effects possibly resulted from the moderate enhancement of osmotic stress in the medium and stronger chaotrope-like behavior from higher molecular weight PEG. With further verification that 0.5 % (w/v) PEG-20000 enabled to reduce the effect of oxygen inhibition on microalgal growth, the PEG-20000-mediated cultivation offers a feasible means for mass culture of N. oculata in closed setting.     

Optical spectroscopic differentiation of various equilibrium denatured states of horse cytochrome c

Thermal unfolding of cytochrome c (cyt c) from several states has been studied using equilibrium spectroscopic techniques. CD in the uv, vibrational circular dichroism, infrared, and uv-vis absorption spectra measured at various temperatures, pHs, salt concentrations, and GuHCl concentrations are used to show the conformational as well as heme structural differences between native and various denatured states. The difference in thermal denaturation behaviors of cyt c starting from acid denatured, molten globule (MG), and the A and native states are explored. Different final high temperature states were observed for cytochrome c unfolding from four different initial states (native, MG, A, and acid denatured state) by electronic CD, Fourier transform infrared (FTIR), and vibrational CD (VCD). Consistent with this, different thermal unfolding pathways for the MG and A states are suggested by the FTIR and VCD data for this process.     

Structural characteristics of thermostable immunogenic outer membrane protein from Salmonella enterica serovar Typhi

In this work, we explored the acid-induced unfolding pathway of non-porin outer membrane protein (OMP), an immunogenic protein from Salmonella Typhi, by monitoring the conformational changes over a pH range of 1.0–7.0 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, and dynamic light scattering. The spectroscopic measurements showed that OMP in its native state at pH 7.0 exists in more stable and compact conformation. In contrast, at pH 2.0, OMP retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii, and nearly four-fold increase in ANS fluorescence with respect to the native state, indicating that MG state exists at pH 2.0. Quenching of tryptophan fluorescence by acrylamide further confirmed the accumulation of a partially unfolded state between native and unfolded state. The effect of pH on the conformation and thermostability of OMP points towards its heat resistance at neutral pH (T _m?~?69 °C at pH 7.0, monitored by change in MRE_{222 nm}). Acid unfolded state was also characterized by the lack of a cooperative thermal transition. All these results suggested that acid-induced unfolded state of OMP at pH 2.0 represented the molten globule state. The chemical denaturation studies with GuHCl and urea as denaturants showed dissimilar results. The chemical unfolding experiments showed that in both far-UV CD and fluorescence measurements, GuHCl is more efficient than urea. GuHCl is characterized by low C _m (~1 M), while urea is characterized by high C _m (~3 M). The fully unfolded states were reached at 2 M GuHCl and 4 M urea concentration, respectively. This study adds to several key considerations of importance in the development of therapeutic agents against typhoid fever for clinical purposes.     

Amyloid fibril formation by a domain of rat cell adhesion molecule

Amyloid fibrils are protein aggregates implicated in several amyloidotic diseases. Cellular membranes with local decrease in pH and dielectric constant are associated with the amyloid formation. In this study, domain 1 of cell adhesion molecule CD2 (CD2-1) is used for studying amyloid fibril formation in a water/trifluoroethanol (TFE) mixture. CD2-1 is an all beta-sheet protein similar in topology to the amyloidogenic light chain variable domain, which deposits as amyloid in light chain amyloidosis at acidic pH. When incubated at pH 2.0 in the presence of 18% TFE, CD2-1 initiates the process to assemble into amyloid fibrils. It has been shown that TFE induces CD2-1 conformational change with a chemical transition (C(m)) of 18% (v/v). ANS (1-anilinonapthalene-8-sulfonic acid) binding was used to show that the hydrophobic surface becomes exposed under these solvent conditions. Our studies indicate that partial formation of a non-native conformation and the exposure of the hydrophobic interior could be the origins of oligomerization and fibril formation of CD2-1.     

Trifluoroethanol-induced "molten globule" state in stem bromelain

2,2,2-Trifluoroethanol (TFE) denatures proteins but also stabilizes/induces alpha helical conformation in partially/completely unfolded proteins. As reported earlier from this laboratory, stem bromelain is known to exist as a partially folded intermediate (PFI) at pH 2.0. The effect of increasing concentration of TFE on the PFI of bromelain has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino 8-naphthalene sulfonic acid (ANS), and near-UV CD temperature transition. Far-UV CD spectra show considerable accumulation of secondary structure at 70% (v/v) concentration of TFE with spectral features resembling the pH 7.0 preparation. Interestingly the partially folded intermediate regained significant tertiary structure/interactions, with increasing concentration of TFE, and at 60% (v/v) TFE approached almost that of the pseudo native (pH 7.0) state. Further increase to 70% (v/v) TFE, however, resulted in complete loss of tertiary structure/interactions. Studies on tryptophan fluorescence also suggested the induction of some compact structure at 60% (v/v) concentration of TFE. The partially folded intermediate showed enhanced binding of the fluorescent probe (ANS) in the presence of 60% (v/v) TFE. Taken together these observations suggest a "molten globule" state between 60 and 70% (v/v) TFE. Thermal transition studies in the near-UV CD region indicated cooperative transition for PFI in the presence of 60% (v/v) TFE changing to noncooperative transition at 70% (v/v) TFE. This was accompanied by a shift in the midpoint of thermal denaturation (T(m)) from 58 to 51 degrees C. Gradual transition and loss of cooperative thermal unfolding in the 60-70% (v/v) range of TFE also support the existence of the molten globule state.     

Fluoroalcohol-induced modulation of the pathway of amyloid protofibril formation by barstar

To understand how the conformational heterogeneity of protofibrils formed by any protein, as well as the mechanisms of their formation, are modulated by a change in aggregation conditions, we studied the formation of amyloid protofibrils by barstar at low pH by multiple structural probes in the presence of hexafluoroisopropanol (HFIP). In the presence of 10% HFIP, aggregation proceeds with the transient formation of spherical oligomers and leads to the formation of both protofibrils and fibrils. Curly short protofibrils and fibrils are seen to form early during the aggregation reaction, and both are seen to grow gradually in length during the course of the reaction. Atomic force microscopy images reveal that the HFIP-induced protofibrils are long (～300 nm in length), curly, and beaded and appear to be composed primarily of β-sheet bilayers, with heights of ～2.4 nm. The protofibrils formed in the presence of HFIP differ in both their structures and their stabilities from the protofibrils formed either in the absence of alcohol or in the presence of a related alcohol, trifluoroethanol (TFE). Aggregation appears to proceed via an isodesmic polymerization mechanism. Internal structure in the growing aggregates changes in two stages during protofibril formation. In the first stage, an α-helix-rich oligomeric intermediate is formed. In the second stage, the level of β-sheet structure increases at the expense of some α-helical structure. The second stage itself appears to occur in two distinct steps. The creation of thioflavin T binding sites occurs concomitantly with aggregate elongation and is seen to precede the change in secondary structure. The long straight fibrils with characteristic heights of 8-10 nm, which form in the course of the HFIP-induced aggregation reaction, have not been observed to form either in the absence of alcohol or in the presence of TFE.