Formation of β-Lactoglobulin Nanofibrils by Microwave Heating Gives a Peptide Composition Different from Conventional Heating

A novel procedure involving microwave heating (MH) at 80 °C can be used to induce self-assembly of β-lactoglobulin (β-lg) into amyloid-like nanofibrils at low pH. We examined the self-assembly induced by MH, and evaluated structural and compositional differences between MH fibrils and those formed by conventional heating (CH). MH significantly accelerated the self-assembly of β-lg, resulting in fully developed fibrils in ≤2 h. However, longer MH caused irreversible disintegration of fibrils. An increase in the fibril yield was observed during the storage of the 2 h MH sample, which gave a yield similar to that of 16 h CH sample. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that the fibrils formed by the two methods do not show significant differences in their secondary structure components. However, they exhibited differences in surface hydrophobicity, and mass spectrometry showed that the MH fibrils contained larger peptides than CH fibrils, including intact β-lg monomers, providing evidence for a different composition between the MH and CH fibrils, in spite of no observed differences in their morphology. We suggest MH initially accelerates the self-assembly of β-lg due to its nonthermal effects on unfolding, nucleation, and subsequent stacking of β-sheets, rather than promoting partial hydrolysis. Thus, MH fibrils are composed of larger peptides, and the observed higher surface hydrophobicity for the MH fibrils was attributed to the parts of the larger peptides extending out of the core structure of the fibrils.     

Modulation of self-association and subsequent fibril formation in an alanine-rich helical polypeptide

Thermal unfolding, reversible self-association, and irreversible aggregation were investigated for an alanine-rich helical polypeptide, 17-H-6, with sequence [AAAQEAAAAQAAAQAEAAQAAQ] 6. Dynamic light scattering, transmission electron microscopy, and thermal unfolding measurements indicate that 17-H-6 spontaneously and reversibly self-associates at acidic pH and low temperature. The resulting multimers have a compact, globular morphology with an average hydrodynamic radius approximately 10-20 nm and reversibly dissociate to monomers upon an increase to pH 7.4. Both free monomer and 17-H-6 chains within the multimers are alpha-helical and folded at low temperature. Reversible unfolding of the monomer occurs upon heating of solutions at pH 7.4. At pH 2.3, heating first causes incomplete dissociation and unfolding of the constituent chains. Further incubation at elevated temperature induces additional structural and morphological changes and results in fibrils with a beta-sheet 2 degrees structure and a characteristic diameter of 5-10 nm (7 nm mean). The ability to modulate association and aggregation suggests opportunities for this class of polypeptides in nanotechnology and biomedical applications.     

Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn(2+) ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn(2+), dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of beta-sheet structure occurs simultaneously with the appearance of well-defined fibrils.     

Trifluoroethanol-induced unfolding of concanavalin A: equilibrium and time-resolved optical spectroscopic studies

Conformational structure changes in concanavalin A (Con A), a legume lectin protein which is composed of 18 beta-strands, induced by dissolving in 50% trifluoroethanol (TFE) were monitored at neutral and low pH by far- and near-UV circular dichroism (CD), fluorescence, and FTIR under equilibrium conditions. Stopped-flow studies using CD and fluorescence as well as FTIR, at low and high protein concentration, respectively, were carried out to follow the time-dependent conformation changes occurring after rapid mixing of the protein with TFE. Equilibrium CD results show that, upon addition of TFE, low-concentration Con A transforms to a highly alpha-helical conformation at both neutral and low pH. However, at neutral pH under high protein concentration conditions, aggregation and precipitation are eventually detected with FTIR, indicating that a final beta-structure is attained. Stopped-flow fluorescence shows the existence of an unfolding intermediate for pH 2.0 and 4.5, which could be related to the dissociation of the dimer form. However, evidence for an intermediate is not obtained at pH 6.7, where the native protein is a tetramer. Stopped-flow FTIR is consistent with these results, indicating formation of a H(+)-stabilized intermediate alpha-helical conformation before aggregation develops. Con A in TFE provides an example of an intermediate with non-native secondary structure appearing on the unfolding pathway. On the basis of the kinetic results obtained, an unfolding mechanism is proposed and some stable intermediates are identified. In turn, the slow structural change of Con A induced by TFE provides a useful model system for study of protein unfolding due to its accessibility with several spectroscopic and kinetic tools.     

Quaternary conformational stability: The effect of reversible self‐association on the fibrillation of two insulin analogs

Under conditions relevant to the manufacturing of insulin (e.g., pH 3, room temperature), biosynthetic human insulin (BHI), and Lispro insulin (Lispro) require a nucleation step to initiate aggregation. However, upon seeding with preformed aggregates, both insulins rapidly aggregate into nonnative fibrils. Far ultraviolet circular dichroism (far‐UV CD) and second derivative Fourier transform infrared (2D‐FTIR) spectroscopic analyses show that the fibrillation process involves a change in protein secondary structure from α‐helical in native insulin to predominantly β‐sheet in the nonnative fibrils. After seeding, Lispro aggregates faster than BHI, likely because of a reduced propensity to reversibly self‐associate. Composition gradient multi‐angle light scattering (CG‐MALS) analyses show that Lispro is more monomeric than BHI, whereas their conformational stabilities measured by denaturant‐induced unfolding are statistically indistinguishable. For both BHI and Lispro, as the protein concentration increases, the apparent first‐order rate constant for soluble protein loss decreases. To explain these phenomena, we propose an aggregation model that assumes fibril growth through monomer addition with competitive inhibition by insulin dimers. Biotechnol. Bioeng. 2011;108: 2359–2370. © 2011 Wiley Periodicals, Inc.     

Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain

The formation of amyloid fibrils by the SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in beta-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis.     

Helix-turn-helix peptides that form alpha-helical fibrils: turn sequences drive fibril structure

Models of apolipoprotein A-I (apo A-I), the main protein of high-density lipoprotein, predict that it contains 10 amphiphilic, alpha-helical segments connected by turns. We synthesized four peptides with two identical 18-residue, amphiphilic, alpha-helical segments (Anantharamaiah, G. M., et al. (1985) J. Biol. Chem. 260, 10248-10255) connected by putative turn sequences from apo A-I: (1) Ac-DWLKAFYDKVAEKLKEAFKVEPLRADWLKAFYDKVAEKLKEAF-NH2, (2) Ac-DWLKAFYDKVAEKLKEAFGLLPVLEDWLKAFYDKVAEKLKEAF-NH2, (3) Ac-DWLKAFYDKVAEKLKEAFKVQPYLDDWLKAFYDKVAEKLKEAF-NH2, and (4) Ac-DWLKAFYDKVAEKLKEAFNGGARLADWLKAFYDKVAEKLKEAF-NH2. Surprisingly, peptides 1-3 formed fibrils after incubation (37 degrees C, 10 mM sodium phosphate, pH 7.60), but in contrast to beta-sheet amyloid fibrils, these did not bind thioflavin T and they induced a blue shift in the spectrum of Congo red. CD (peptides 1-3) and FTIR (peptides 1 and 2) of the fibrils showed significant alpha-helical character. Synchrotron X-ray fiber diffraction on a magnetically aligned sample of 1 confirmed the alpha-helical character in the fibrils and indicated that the helical axes are oriented perpendicular to the fibril axis. In contrast, peptide 4, containing two Gly residues but no Pro in the turn, formed only a small amount of nonfibrillar precipitate after prolonged incubation. Peptide 4P (peptide 4 with a Pro in place of the central Ala) and peptide 5, containing a PEG block in lieu of the central turn, were similar to peptide 4 in not forming fibrils, possibly because the region linking the helices was unstructured. These studies indicate that varying turn sequences between longer amphiphilic alpha-helical segments can drive the structure of fibrils.     

Conformational and aggregation properties of a PEGylated alanine-rich polypeptide

The conformational and aggregation behavior of PEG conjugates of an alanine-rich polypeptide (PEG-c17H6) were investigated and compared to that of the polypeptide equipped with a deca-histidine tag (17H6). These polypeptides serve as simple and stimuli-responsive models for the aggregation behavior of helix-rich proteins, as our previous studies have shown that the helical 17H6 self-associates at acidic pH and converts to β-sheet structures at elevated temperature under acidic conditions. In the work here, we show that PEG-c17H6 also adopts a helical structure at ambient/subambient temperatures, at both neutral and acidic pH. The thermal denaturation behavior of 17H6 and PEG-c17H6 is similar at neutral pH, where the alanine-rich domain has no self-association tendency. At acidic pH and elevated temperature, however, PEGylation slows β-sheet formation of c17H6, and reduces the apparent cooperativity of thermally induced unfolding. Transmission electron microscopy of PEG-c17H6 conjugates incubated at elevated temperatures showed fibrils with widths of ～20-30 nm, wider than those observed for fibrils of 17H6. These results suggest that PEGylation reduces β-sheet aggregation in these polypeptides by interfering, only after unfolding of the native helical structure, with interprotein conformational changes needed to form β-sheet aggregates.     

Analysis of the conformation and stability of human bronchial lysozyme by circular dichroism.

1. Human bronchial lysozyme was isolated from nonpurulent secretions and studied by circular dichroism (CD) spectroscopy for its conformational properties. 2. The two negative bands at 208 and 222 nm indicated that the peptide chain adopted an alpha-helical structure in physiological conditions. 3. The molecule was stable at pH 1.0 but not at pH 12.0. 4. Increasing ionic strength by adding NaCl up to 1 M did not change the CD spectra. 5. Complete unfolding of the molecule by guanidinium chloride was obtained only at the concentration of 6 M. 6. Bronchial lysozyme was also denatured by sodium dodecyl sulphate. 7. The molecule was stable when mild reduction was performed at 37 degrees C for 30 min but was completely unfolded after heating at 100 degrees C for 3 min.     

A pH-dependent conformational change in EspA, a component of the Escherichia coli O157:H7 type III secretion system

pH-Dependent structural changes for Escherichia coli O157:H7 EspA were characterized by CD, 8-anilino-2-naphthyl sulfonic acid (ANS) fluorescence, and sedimentation equilibrium ultracentrifugation. Far- and near-UV CD spectra, recorded between pH 2.0 and 7.0, indicate that the protein has significant amounts of secondary and tertiary structures. An increase in ANS fluorescence intensity (in the presence of EspA) was observed at acidic pH; whereas, no increased ANS fluorescence was observed at pH 7.0. These results suggest the presence of a partially unfolded state. Interestingly, urea-induced unfolding transitions, monitored by far-UV CD spectroscopy, showed that the protein is destabilized at pH 2.0 as compared with EspA at neutral pH. Although increased ANS fluorescence was observed at pH 3.0, the urea-induced unfolding curve is similar to that found at pH 7.0. This result suggests the presence, at pH 3.0, of an ordered, but partially unfolded state, which differs from typical molten globule. The results of analytical ultracentrifugation and infrared spectroscopy indicate that EspA molecules associate at pH 7.0, suggesting the formation of short filamentous oligomers containing alpha-helical structures, whereas the protein tend to form nonspecific aggregates containing intermolecular beta-sheets at pH 2.0. Our experiments indicate that EspA has the potential to spontaneously form filamentous oligomers at neutral pH; whereas the protein is partially unfolded, assuming different conformations, at acidic pH.     

Surface denaturation and amyloid fibril formation of insulin at model lipid-water interfaces

We consider the effects that different lipid surfaces have upon the denaturation and subsequent formation of amyloid fibrils of bovine insulin. The adsorption and unfolding kinetics of insulin being adsorbed onto the different lipid surfaces under denaturing conditions are studied using FTIR ATR spectroscopy and are compared to the bulk solution behavior of the protein. Atomic force microscopy studies are also performed to compare the fibrils growing on the different surfaces. This study shows that both the adsorption and unfolding kinetics of insulin can be described by a sum of exponential processes and that different surfaces behave differently, with respect both to one another and to the bulk protein solution. The proteins adsorbed onto the surfaces are observed to have faster unfolding kinetics than those in the bulk, and the fibril-like structures formed at the surfaces are shown to be different in a number of ways from those found in bulk solution. The beta-sheet content and growth kinetics of the adsorbed proteins also differ from those of the bulk system. An attempt is made to describe the observed behavior in terms of simple physical arguments involving adsorption, unfolding, and aggregation of the proteins.     

Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase

Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30 degrees C). A plot of temperature-induced changes in (1)H-(2)H exchange, the decrease in the absorbance of the alpha-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45 degrees C and the major one around 59 degrees C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52 degrees C, respectively). The transition, which occurs at 39-45 degrees C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52-59 degrees C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7 degrees C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS.     

The enhancing effect of homocysteine thiolactone on insulin fibrillation and cytotoxicity of insulin fibril

In the current study both structural alteration and fibrillation of insulin were studied in the presence of homocysteine thiolactone (HCTL). The spectroscopic studies revealed that HCTL increases rate of insulin unfolding, giving rise to the appearance of solvent-exposed hydrophobic regions and induces a transition from α-helix into predominantly β-sheet structures. Thioflavin-T fluorescence studies revealed that HCTL markedly enhanced the quantity of insulin fibril formation in both agitating and non-agitating systems. Also gel electrophoresis results suggest that HCTL accelerates the process of formation of high molecular weight insulin aggregates. Moreover, insulin fibrils obtained in the presence of HCTL and those collected earlier in the pathway of insulin fibrillation displayed improved cytotoxicity against cancer cells. The enhancement of insulin fibril formation with elevated cytotoxic properties as occurred in the presence of HCTL, may suggest this homocysteine derivative as a possible contributing factor in the pathology of insulin fibrils.     

Thermally induced fibrillar aggregation of hen egg white lysozyme

We study the effect of pH and temperature on fibril formation from hen egg white lysozyme. Fibril formation is promoted by low pH and temperatures close to the midpoint temperature for protein unfolding (detected using far-ultraviolet circular dichroism). At the optimal conditions for fibril formation (pH 2.0, T = 57 degrees C), on-line static light-scattering shows the formation of fibrils after a concentration-independent lag time of approximately 48 h. Nucleation presumably involves a change in the conformation of individual lysozyme molecules. Indeed, long-term circular dichroism measurements at pH 2.0, T = 57 degrees C show a marked change of the secondary structure of lysozyme molecules after approximately 48 h of heating. From atomic force microscopy we find that most of the fibrils have a thickness of approximately 4 nm. These fibrils have a coiled structure with a periodicity of approximately 30 nm and show characteristic defects after every four or five turns.     

Persistence of tertiary structure in 7.9 M guanidinium chloride: the case of endo-beta-1,3-glucanase from Pyrococcus furiosus

The Pyrococcus furiosus endo-beta-1,3-glucanase belongs to the subfamily of laminarinase, which can be classified as "all beta proteins" as confirmed by deconvolution of far-UV CD and FTIR spectra. The persistence of a significant amount of tertiary structure in 7.9 M GdmCl, as indicated by near-UV CD spectroscopy, accompanied by a red-shift of the maximum fluorescence emission wavelength is a peculiar property of this hyperthermophilic endoglucanase. The possibility to observe tertiary structure elements under extremely denaturing conditions is notable and is limited to only a few examples. The unusual resistance toward guanidinium chloride denaturation is paralleled by a notable stability at extremely low pH and at high temperature. The analysis of the protein spectral properties indicates that the secondary structure elements are preserved down to pH 1.0 and up to 90 degrees C at pH 7.4 and pH 3.0. The study of the conditions that determine the persistence of residual structure at high denaturant concentration and the examination of these structures are particularly interesting because these state(s) may be preliminary or coincident with the coalescence of protein aggregates or to the formation of amyloid-like fibrils, and they may serve as seeds of protein folding.     

Stimulation and inhibition of fibril formation by a peptide in the presence of different concentrations of SDS

Sodium dodecyl sulphate (SDS), a detergent that mimics some characteristics of biological membranes, has been found to affect significantly fibril formation by a peptide from human complement receptor 1. In aqueous solution the peptide is unfolded but slowly aggregates to form fibrils. In sub-micellar concentrations of SDS the peptide is initially alpha-helical but converts rapidly to a beta-sheet structure and large quantities of fibrils form. In SDS above the critical micellar concentration the peptide adopts a stable alpha-helical structure and no fibrils are observed. These findings demonstrate the sensitivity of fibril formation to solution conditions and suggest a possible role for membrane components in amyloid fibril formation in living systems.     

The presence of non‐native helical structure in the unfolding of a beta‐sheet protein MPT63

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β‐sandwich protein containing 11 β strands and a very short stretch of 3₁₀ helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β‐sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV‐CD and FTIR spectra clearly suggest that the low‐pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β‐sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non‐native helical propensity of the protein structure.     

Secondary structures of a new class of lipid body proteins from oilseeds.

The three main isoforms of the 19-kDa lipid body proteins (oleosin) have been purified to homogeneity from embryos of rapeseed. The secondary structures of these proteins as derived from circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy were compared with the secondary structures predicted from the primary sequences. The salient feature of the primary sequence of all oleosins is its division into three defined structural domains: a central hydrophobic domain flanked on either side by relatively hydrophilic domains, respectively. Using a variety of predictive methods based on primary amino acid sequence data, the oleosins exhibited a high probability of beta-strand structure in the 70-residue central hydrophobic domain, with relatively little alpha-helical content. Secondary structure data derived from CD and FTIR were consistent with the predictions from primary sequence, showing that the oleosins contained about 45% beta-strand and 13% alpha-helical structure. Under high salt conditions, a 40-kDa polypeptide was obtained from purified preparations of the 19-kDa oleosins. The 40-kDa polypeptide has a very similar secondary structure, as analyzed by CD and FTIR, to that of the 19-kDa oleosins. This polypeptide is therefore probably a dimer of the 19-kDa oleosins that is formed in high salt environments. A model of the general structure of oleosins is proposed whereby the central hydrophobic domain of the protein with a predominantly beta-strand structure is embedded into the non-aqueous phase of lipid-bodies. This hydrophobic region is flanked by putative alpha-helical structures in the polar N- and C-terminal domains which are probably oriented at the lipid-water interface.     

Spectroscopic investigation on gel-forming beta-sheet assemblage of peptide derivatives

The conformational studies of peptide derivatives A and B in a gel state were studied by using circular dichroism (CD), Fourier transformed infrared (FTIR), and fluorescence spectroscopic techniques. Birefringence and electron microscopic studies were carried out to characterize the morphological aspects of the fibrils in the gel. The FTIR spectra of the peptides show the absence of free NH in the gel state, implying that the intermolecular hydrogen-bond formation is the driving force for the aggregation. The CD spectrum of the peptide gels shows the presence of antiparallel and parallel beta-sheet conformation for peptide derivatives A and B, respectively. Electron microscopic studies (EM) of the peptide derivatives A and B reveal that peptide A formed rigid, rod-like structures without cross-linking and peptide B formed loose fibrils organized into highly noncovalently cross-linked mesh-like structural aggregates. Peptide A was much more soluble in alcoholic solvents than peptide B, and no birefringence was observed with Congo red (CR) staining in the temperature range of 0-80 degrees C. The spectroscopic studies indicate that peptide B consists of domains having a significant amount of beta-sheet structure and exhibiting golden yellow birefringence between 53 and 56 degrees C when stained with Congo red. On the other hand, peptide A gives no evidence of birefringence under polarized light. Fluorescence probe binding studies with pyrene in gel state with peptides A and B indicates the polarity in the interior of the aggregates. The data presented in the present work indicate that peptide B forms fibrils, which is similar to amyloid aggregates that are present in biological systems.     

Equilibrium unfolding CD studies of bovine beta-lactoglobulin and its 14-52 fragment at acidic pH

Bovine beta-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this beta-barrel protein has a surprisingly high alpha-helical preference, which was retained for short fragments. Cooperative transitions from the native beta-sheet to alpha-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high alpha-helical preference, it has been proposed that non-native alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37 degrees C, revealed an apparent two-state transition, and afforded no evidence of non native alpha-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the beta to alpha structure, provoked by organic solvents in the intact protein.