Among various alcohols, those substituted with fluorine, such as 2,2,2-trifluoroethanol (TFE) or 3,3,3,3‘,3‘,3‘-hexafluoro-2-propanol (HFIP), have a marked potential to induce the formation of α-helical structures in peptides and to denature the native structures of proteins. However, the mechanism by which these alcohols exert their effects is unknown. Melittin, a bee venom peptide, is unfolded in the absence of alcohol, but is transformed to an α-helical structure upon addition of alcohols. On the other hand, addition of alcohols to β-lactoglobulin, a predominantly β-sheet protein, denatures the molecule and transforms it to an α-helical structure. We examined the role of several factors in these alcohol-induced transitions, i.e., relative dielectric constant, strength of hydrogen bond estimated by the pH titration of salicylic acid, and clustering of alcohol molecules measured by solution X-ray scattering. Although relative dielectric constant and hydrogen bond strength were confirmed to be important, they did not explain the marked effects of TFE and HFIP. X-ray scattering detected clusters of TFE or HFIP molecules in alcohol/water mixtures with a maximum at around 30% (v/v) of each alcohol. When the conformational transitions induced by TFE and HFIP were plotted against the extent of cluster formation by the corresponding alcohol/water mixtures, the TFE and HFIP-induced transition curves agreed with each other for both melittin and β-lactoglobulin. This suggests that clustering of alcohol molecules is an important factor that enhances the effects of alcohols on proteins and peptides.

Insulin has a largely ␣-helical structure and exists as a mixture of hexameric, dimeric, and monomeric states in solution, depending on the conditions: the protein is monomeric in 20% acetic acid. Insulin forms amyloid-like fibrils under a variety of conditions, especially at low pH. In this study we investigated the fibrillation of monomeric human insulin by monitoring changes in CD, attenuated total reflectance-Fourier transform infrared spectroscopy, 8-anilinonaphthalenesulfonic acid fluorescence, thioflavin T fluorescence, dynamic light scattering, and H/D exchange during the initial stages of the fibrillation process to provide insight into early events involving the monomer. The results demonstrate the existence of structural changes occurring before the onset of fibril formation, which are detectable by multiple probes. The data indicate at least two major populations of oligomeric intermediates between the native monomer and fibrils. Both have significantly non-native conformations, and indicate that fibrillation occurs from a betarich structure significantly distinct from the native fold.A number of human diseases are caused by the pathogenic deposition of proteins in the form of amyloid-like fibrils (1-7). Several non-pathogenic proteins and peptides also undergo amyloid like fibril formation on destabilization of their native state (7-10). The fact that structurally and sequentially non-homologous proteins are able to self-assemble into fibrils possessing similar morphology (e.g. 10 -18 nm width, birefringence to polarized light, and cross-␤ structure) suggests a common molecular mechanism in the fibrillation pathways. A variety of hypotheses for the mechanism of fibril formation have been proposed.Insulin is a 51-residue hormone with a largely ␣-helical structure. It exists as a mixture of hexameric, dimeric, and monomeric states in solution, with the relative population of different oligomeric species being strongly dependent on the environmental conditions: the protein is predominantly monomeric in 20% acetic acid, dimeric in 20 mM HCl, and hexameric at pH 7.5 in the presence of zinc. Insulin forms amyloidlike fibrils under a variety of conditions (11-13), with various overall morphologies depending on the arrangement of constituent protofilaments (14, 15). Insulin fibrils pose a variety of problems in biomedical and biotechnological applications. Amyloid deposits of insulin have been observed in patients with diabetes after repeated injection and in normal aging, as well as after subcutaneous insulin infusion (16,17).In our previous work we have shown the important role of partially folded intermediates in insulin fibrillation in vitro (13, 18 -20). In fact, our studies showed that insulin, which is a hexamer at physiological pH, undergoes rapid fibrillation starting at relatively low concentrations of guanidine hydrochloride and urea. The predominant species characterized by various biophysical techniques under these conditions was shown to be a partially folded, expanded monomer, which is presen...

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The flavonoid baicalein inhibits fibrillation of α-synuclein, which is a major component of the Lewy bodies in Parkinson's disease. It has been known that baicalein induces the formation of the α-synuclein oligomers and consequently prevents its fibrillation. In order to evaluate structural properties of the baicalein-stabilized oligomers, we purified the oligomer species by HPLC and examined their stability and structure by CD, FTIR, SEC-HPLC, SAXS, and AFM. Baicaleinstabilized oligomers are β-sheet-enriched according to CD and FTIR analysis. They did not form fibrils even after very prolonged incubation. From the SAXS data and AFM images, the oligomers were characterized as quite compact globular species. Oligomers were extremely stable, with a GdmCl C m =3.3 M. This high stability explains the previously observed inhibition properties of baicalein toward α-synuclein fibrillation. These baicalein-stabilized oligomers, being added to the solution of the aggregating α-synuclein, were able to noticeably inhibit its fibrillation. After the prolonged co-incubation, short fibrils were formed, suggesting effective interaction of oligomers with the monomeric α-synuclein. Membrane permeability tests suggested that the baicalein-stabilized oligomers had a mild effect on the integrity of the membrane surface. This effect was rather similar to that of the monomeric protein, suggesting that the targeted stabilization of certain α-synuclein oligomers might offer a potential strategy for the development of novel Parkinson's disease therapies.

The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.