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.
Despite numerous efforts, the lack of detailed structural information on amyloid fibrils has hindered clarification of the mechanism of their formation. Here, we describe a novel procedure for characterizing the conformational flexibility of beta(2)-microglobulin amyloid fibrils at single-residue resolution that uses H/D exchange of amide protons combined with NMR analysis. The results indicate that most residues in the middle region of the molecule, including the loop regions in the native structure, form a rigid beta-sheet core, whereas the the N- and C-termini are excluded from this core. The extensively hydrogen-bonded beta-sheet core explains the remarkable rigidity and stability of amyloid fibrils. The present method could be used to obtain residue-specific conformational information of various amyloid fibrils, even though it does not provide a high resolution three-dimensional structure.
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