Hexafluoroisopropanol induces amyloid fibrils of islet amyloid polypeptide by enhancing both hydrophobic and electrostatic interactions

Although amyloid fibrils deposit with various proteins, the comprehensive mechanism by which they form remains unclear. We studied the formation of fibrils of human islet amyloid polypeptide associated with type II diabetes in the presence of various concentrations of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) under acidic and neutral pH conditions using CD, amyloid-specific thioflavin T fluorescence, fluorescence imaging with thioflavin T, and atomic force microscopy. At low pH, the formation of fibrils was promoted by HFIP with an optimum at 5% (v/v). At neutral pH in the absence of HFIP, significant amounts of amorphous aggregates formed in addition to the fibrils. The addition of HFIP suppressed the formation of amorphous aggregates, leading to a predominance of fibrils with an optimum effect at 25% (v/v). Under both conditions, higher concentrations of HFIP dissolved the fibrils and stabilized the α-helical structure. The results indicate that fibrils and amorphous aggregates are different types of precipitates formed by exclusion from water-HFIP mixtures. The exclusion occurs through the combined effects of hydrophobic interactions and electrostatic interactions, both of which are strengthened by low concentrations of HFIP, and a subtle balance between the two types of interactions determines whether the fibrils or amorphous aggregates dominate. We suggest a general view of how the structure of precipitates varies dramatically from single crystals to amyloid fibrils and amorphous aggregates.     

Dehydration stability of amyloid fibrils studied by AFM

Atomic force microscopy was used to investigate the stability of dehydrated amyloid fibrils formed by human islet polypeptide (IAPP) and Aβ(1–42) peptides. IAPP amyloid fibrils were imaged in liquid (hydrated state) and in air (dehydrated). In addition, fibrils dried on the mica surface were rehydrated and re-examined both in liquid and in air (after consecutive redrying). As reported previously, the initial drying process does not result in any major change in the amyloid appearance and the dimensions of the fibrils are preserved. However, when once-dried samples are rehydrated, fibril stability is lost. The fibrils disintegrate into small particles that are attached to the mica surface. This process is further confirmed by studies of the rehydrated samples after drying, on which the morphology of the fibrils is clearly changed. Similar behavior is observed for Aβ(1–42) amyloid fibrils, which are apparently stable on first drying, but disintegrate on rehydration. The observed change indicates that dehydration is causing a change in the internal structure of the amyloid fibrils. This has important implications for studies of amyloid fibrils by other techniques. Due to the potential influence of hydration and sample history on amyloid structure, preparation and study of amyloid samples with controlled humidity requires more consideration.     

The relative contribution of electrostatic interactions to stabilization of collagen fibrils

Electrostatic energies of interaction between type I collagen molecules were calculated, using models developed by Timasheff and Hill. These energies, along with a contribution from hydrophobic forces, were then incorporated into an equation due to Flory that described phase equilibria of rod-like polymers. The Flory formalism in turn permitted a calculation of the overall free energy of fibril formation (delta Ff), and an assessment of the relative contribution of electrostatic and hydrophobic forces to delta Ff. Lastly, delta Ff was used in a nucleation-growth model relating halftimes of fibril formation (t1/2) to ionic strength (I) and temperature. Because the theory provided no basis for setting absolute levels of the energetic contributions, five parameters in the model had to be derived from experimental data. Based on the fit of theory to experimental results both for intact and pepsinized collagen, it was found that very low electrostatic energies (about -1 kcal/mole per collagen molecule) were sufficient to explain experimental t1/2 vs I relationships. This energy is equivalent to 1 close charge-pair interaction per molecule and appears to be lower than the energy assignable to hydrophobic interactions.     

Nature and significance of the interactions between amyloid fibrils and biological polyelectrolytes

Charged polyelectrolytes such as glycosaminoglycans and nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases. We have investigated the nature and generality of this phenomenon by studying the ability of different polyanions, including DNA, ATP, heparin, and heparan sulfate, to promote the aggregation of amyloidogenic proteins and to bind to the resulting aggregates. Preformed amyloid fibrils of human muscle acylphosphatase and human lysozyme, proteins with a net positive charge at physiological pH values, were found to bind tightly to the negatively charged DNA or ATP. The effects of the polyelectrolytes on the kinetics of aggregation were studied for acylphosphatase, and the presence of ATP, DNA, or heparin was found to increase its aggregation rate dramatically, with a degree dependent on the net charge and size of the polyanion. Magnesium or calcium ions were found to attenuate, and ultimately to suppress, these interactions, suggesting that they are electrostatic in nature. Moreover, heparin was found to stabilize the aggregated state of acylphosphatase through compensation of electrostatic repulsion. Noteworthy, differences in affinity between native and aggregated acylphosphatase with heparin suggest that amyloid fibrils can themselves behave as polyelectrolytes, interacting very strongly with other polyelectrolytes bearing the opposite charge. Within an in vivo context, the strengthening of the electrostatic interactions with other biological polyelectrolytes, as a consequence of protein misfolding and aggregation, could therefore result in depletion of essential molecular components and contribute to the known cytotoxicity of amyloid fibrils and their precursors.     

Plasticity of amyloid fibrils

In experiments designed to characterize the basis of amyloid fibril stability through mutational analysis of the Abeta (1-40) molecule, fibrils exhibit consistent, significant structural malleability. In these results, and in other properties, amyloid fibrils appear to more resemble plastic materials generated from synthetic polymers than globular proteins. Thus, like synthetic polymers and plastics, amyloid fibrils exhibit both polymorphism, the ability of one polypeptide to form aggregates of different morphologies, and isomorphism, the ability of different polypeptides to grow into a fibrillar amyloid morphology. This view links amyloid with the prehistorical and 20th century use of proteins as starting materials to make films, fibers, and plastics, and with the classic protein fiber stretching experiments of the Astbury group. Viewing amyloids from the point of view of the polymer chemist may shed new light on a number of issues, such as the role of protofibrils in the mechanism of amyloid formation, the biological potency of fibrils, and the prospects for discovering inhibitors of amyloid fibril formation.     

Positional effects of phosphorylation on the stability and morphology of tau-related amyloid fibrils

Hyperphosphorylated forms of tau protein are the main component of paired helical filaments (PHFs) of neurofibrillary tangles in the brain of Alzheimer's disease patients. To understand the effect of phosphorylation on the fibrillation of tau, we utilized tau-derived phosphorylated peptides. The V(306)QIVYK(311) sequence (PHF6) in the microtubule-binding domain is known to play a key role in the fibrillation of tau, and the short peptide corresponding to the PHF6 sequence forms amyloid-type fibrils similar to those generated by full-length tau. We focused on the amino acid residue located at the N-terminus of the PHF6 sequence, serine or lysine in the native isoform of tau, and synthesized the PHF6 derivative peptides with serine or lysine at the N-terminus of PHF6. Peptides phosphorylated at serine and/or tyrosine were synthesized to mimic the possible phosphorylation at these positions. The critical concentrations of the fibrillation of peptides were determined to quantitatively assess fibril stability. The peptide with the net charge of near zero tended to form stable fibrils. Interestingly, the peptide phosphorylated at the N-terminal serine residue exhibited remarkably low fibrillation propensity as compared to the peptide possessing the same net charge. Transmission electron microscopy measurements of the fibrils visualized the paired helical or straight fibers and segregated masses of the fibers or heterogeneous rodlike fibers depending on the phosphorylation status. Further analyses of the fibrils by the X-ray fiber diffraction method and Fourier transform infrared spectroscopic measurements indicated that all the peptides shared a common cross-β structure. In addition, the phosphoserine-containing peptides showed the characteristics of β-sandwiches that could interact with both faces of the β-sheet. On the basis of these observations, possible protofilament models with four β-sheets were constructed to consider the positional effects of the serine and/or tyrosine phosphorylations. The electrostatic intersheet interaction between phosphate groups and the amino group of lysine enhanced the lateral association between β-sheets to compensate for the excess charge. In addition to the previously postulated net charge of the peptide, the position of the charged residue plays a critical role in the amyloid fibrillation of tau.     

Hydrophobic, aromatic, and electrostatic interactions play a central role in amyloid fibril formation and stability

Amyloid-like fibrous crystals formed by the peptide KFFEAAAKKFFE have been previously characterized and provide an ideal model system to examine the importance of specific interactions by introducing specific substitutions. We find that the removal of any phenylalanine residue completely abrogates assembly ability, while charged residues modulate interactions within the structure resulting in alternative fibrillar morphologies. X-ray fiber diffraction analysis reveals that the essential backbone packing of the peptide molecules is maintained, while small changes accommodate differences in side chain size in the variants. We conclude that even very short peptides are adaptable and add to the growing knowledge regarding amyloid polymorphisms. Additionally, this work impacts on our understanding of the importance of residue composition for amyloidogenic peptides, in particular the roles of electrostatic, aromatic, and hydrophobic interactions in amyloid assembly.     

Critical balance of electrostatic and hydrophobic interactions is required for beta 2-microglobulin amyloid fibril growth and stability

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.     

Powering DNA repair through substrate electrostatic interactions

The reaction catalyzed by the DNA repair enzyme uracil DNA glycosylase (UDG) proceeds through an unprecedented stepwise mechanism involving a positively charged oxacarbenium ion sugar and uracil anion leaving group. Here we use a novel approach to evaluate the catalytic contribution of electrostatic interactions between four essential phosphodiester groups of the DNA substrate and the cationic transition state. Our strategy was to substitute each of these phosphate groups with an uncharged (R)- or (S)-methylphosphonate linkage (MeP). We then compared the damaging effects of these methylphosphonate substitutions on catalysis with their damaging effects on binding of a cationic 1-azadeoxyribose (1-aza-dR(+)) oxacarbenium ion analogue to the UDG-uracil anion binary complex. A plot of log k(cat)/K(m) for the series of MeP-substituted substrates against log K(D) for binding of the 1-aza-dR(+) inhibitors gives a linear correlation of unit slope, confirming that the electronic features of the transition state resemble that of the 1-aza-dR(+), and that the anionic backbone of DNA is used in transition state stabilization. We estimate that all of the combined phosphodiester interactions with the substrate contribute 6-8 kcal/mol toward lowering the activation barrier, a stabilization that is significant compared to the 16 kcal/mol catalytic power of UDG. However, unlike groups of the enzyme that selectively stabilize the charged transition state by an estimated 7 kcal/mol, these phosphodiester groups also interact strongly in the ground state. To our knowledge, these results provide the first experimental evidence for electrostatic stabilization of a charged enzymatic transition state and intermediate using the anionic backbone of DNA.     

Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size

Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-β-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions—perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including αB-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.     

Probing the pressure-temperature stability of amyloid fibrils provides new insights into their molecular properties

A number of medical disorders, including Alzheimer's disease and type II diabetes, is characterised by the deposition of amyloid fibrils in tissue. The insolubility and size of the fibrils has largely precluded the determination of their structures at high resolution. Studies probing the stability of amyloid fibrils can reveal which non-covalent interactions are important in the formation and maintenance of the fibril structure. In particular, we review here the use of high hydrostatic pressure and high temperature as perturbation techniques. In general, small aggregates formed early in the assembly process can be dissociated by high pressure, but mature amyloid fibrils are highly pressure stable. This finding suggests that a temporal transition occurs during which side chain packing and hydrogen bond formation are optimised, whereas the hydrophobic effect and electrostatic interactions play a dominant role in the early stages of the aggregation. High temperatures, however, can disrupt most aggregates. Though the observed stability of amyloid fibrils is not unique to these structures, the notion that amyloid fibrils can represent the global minimum in free energy is supported by this type of investigations. Some implications regarding the nature of toxic species, associated with at least many of the amyloid disorders, and recently proposed structural models are discussed.     

Structure-activity relationship of amyloid fibrils

Protein aggregation is a process in which proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as either amorphous or highly ordered, the most common form of the latter being amyloid fibrils. Amyloid fibrils composed of cross-β-sheet structure are the pathological hallmarks of several diseases including Alzheimer’s disease, but are also associated with functional states such as the fungal HET-s prion. This review aims to summarize the recent high-resolution structural studies of amyloid fibrils in light of their (potential) activities. We propose that the repetitive nature of the cross-β-sheet structure of amyloids is key for their multiple properties: the repeating motifs can translate a rather non-specific interaction into a specific one through cooperativity.     

Surface electrostatic interactions contribute little of stability of barnase

Electrostatic interactions are believed to play an important role in stabilizing the native structure of proteins. We have quantified the contribution to stability of an interaction between two oppositely charged side-chains on the surface of barnase. Using site-directed mutagenesis, glutamate 28 and lysine 32 were introduced onto the solvent-accessible side of the second alpha-helix in barnase. These two residues are separated by one turn of the helix, and so are ideally situated for their opposite charges to interact. Double mutant cycle analysis reveals that the interaction between Glu28 and Lys32 contributes only approximately 0.2 kcal/mol to stability of the protein. All other interactions between exposed charged side-chains in barnase examined so far also contribute little to stability. We explain this low value by their location on the surface, rather than in the interior, of the protein.     

Conformational stability of amyloid fibrils of beta2-microglobulin probed by guanidine-hydrochloride-induced unfolding

Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. Beta2-microglobulin (beta2-m) is a major component of the amyloid fibrils observed in patients with dialysis-related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of beta2-m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils.     

Cytotoxicity of amyloid fibrils of X-protein

It is known that amyloid oligomers, protofibrils, and fibrils induce cell death, and antibiotic tetracycline inhibits the fibrillization of beta amyloid peptides and other amyloidogenic proteins and disassembles their pre-formed fibrils. Earlier we have demonstrated that sarcomeric cytoskeletal proteins of the titin family (X-, C-, and H-proteins) are capable to form in vitro amyloid fibrils, and tetracycline effectively destroys these fibrils. Here we show that the viability of polymorphonuclear leukocytes in the presence of X-protein amyloids depends on the concentration of amyloid fibrils of X-protein and the time of incubation. In addition to the disaggregation of X-protein fibrils, tetracycline eliminated the cytotoxic effect of the protein. The antibiotic itself did not show a toxic effect, and the cell viability in its presence even increased. Our results evidence the potential of this approach for evaluating the effectiveness of drugs preventing or treating amyloidoses.     

Charge substitution shows that repulsive electrostatic interactions impede the oligomerization of Alzheimer amyloid peptides

The strong pH dependence of A beta oligomerization could arise from favorable intermolecular charge-charge interactions between His and carboxylate groups, or, alternatively, by mutual electrostatic repulsion of peptide molecules. To test between these two possibilities, the pH dependence of the oligomerization of A beta and three charge substitution variants with Asp, Glu and His substituted by Ala is measured. All four peptides oligomerize, as detected by thioflavin T fluorescence, turbidity, and amyloid fibril formation; therefore, specific charge-charge interactions are nonessential for oligomerization. The strong negative correlation between net charge and oligomerization indicates that electrostatic repulsion between A beta monomers impedes their association.     

Regulation of promoter activity through electrostatic interactions with RNA-polymerase

Electrostatic profiles for sigma70-specific promoters of T4 bacteriophage and four consensus-like synthetic promoters have been calculated. It was shown that the promoters can be classified according to the presence of some specific electrostatic elements located in the far upstream region of their DNAs. A correlation between the electrostatic elements specific for promoters and their functional behaviour was established. A general scheme of promotor activity regulation through electrostatic interactions between the upstream region of promoter DNA and the RNA-polymerase a-subunit was suggested.