Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer's disease beta A4 peptides.

The deposition of amyloid protein aggregates in brain is the main pathological feature of Alzheimer's disease. Their principal constituent is a peptide termed beta A4, which comprises up to 43 amino acid residues. It is highly insoluble under physiological conditions and aggregates into filaments that form very dense clusters in vivo and in vitro. Based on a beta A4 prototype sequence spanning residues 10 to 42 or 43, we have designed analogues in which hydrophobic amino acid residues in position 17 to 20 were substituted by more hydrophilic residues. Depending on the kind of newly introduced amino acids and their position within the sequence, the substitution of only two residues led to variants exhibiting a broad spectrum of different properties. Common to them was a reduced beta-sheet content after solubilization in water and in the solid state. Some of the variants showed significantly reduced amyloidogenicity: although still forming filaments, they did not aggregate into the highly condensed depositions that are typical for amyloid. In addition, they could be solubilized in 200 mM-NaCl and KCl. When mixed with beta A4 peptides bearing the natural sequence, two of the analogues could inhibit the formation of filaments in vitro. These results demonstrate that a well-preserved hydrophobic core around residues 17 to 20 of beta A4 is crucial for the formation of beta-sheet structure and the amyloid properties of beta A4. The introduction of structural alterations within this region may guide the development of reagents for the therapy of Alzheimer's disease.     

Aggregation and neurotoxicity of mutant amyloid beta (A beta) peptides with proline replacement: importance of turn formation at positions 22 and 23

Aggregation of the amyloid beta peptides (A beta 1-42 and A beta 1-40) plays a pivotal role in pathogenesis of Alzheimer's disease. Although it is widely accepted that the aggregates of A betas mainly consist of beta-sheet structure, the precise aggregation mechanism remains unclear. To identify amino acid residues that are important for the beta-sheet formation, a series of proline-substituted mutants of A beta 1-42 peptides at positions 19-26 was synthesized in a highly pure form and their aggregation ability and neurotoxicity on PC12 cells were investigated. All proline-substituted A beta 1-42 mutants except for 22P- and 23P-A beta 1-42 were hard to aggregate and showed weaker cytotoxicity than wild-type A beta 1-42, suggesting that the residues at positions 19-21 and 24-26 are important for the beta-sheet formation. In contrast, 22P-A beta 1-42 extensively aggregated with stronger cytotoxicity than wild-type A beta 1-42. Since proline has a propensity for beta-turn structure as a Pro-X corner, these data implicate that beta-turn formation at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of A beta peptides.     

Human and rodent sequence analogs of Alzheimer's amyloid beta A4 share similar properties and can be solubilized in buffers of pH 7.4

The filamentous amyloid protein aggregates found in the brain of patients affected with Alzheimer's disease principally consist of a peptide termed beta A4, according to its secondary structure of beta-pleated sheets and its molecular mass of about 4 kDa. It has a length of up to 42 or 43 residues. By chemical means, we have synthesized peptide analogs corresponding to the human and rodent beta A4 sequences. We describe structural and functional properties of peptides spanning residues 1-43, 10-23, 1-27 and 4-27 of beta A4. The peptides have been tested for their ability to form filaments in vitro. Their solubilities and secondary structures in solution and in the solid state have been used to detect differences between the properties of human and rodent beta A4 sequences. We show that mouse and rat beta A4 homologs are as amyloidogenic as the human sequence. The absence of amyloid deposits in the brain of aged rats and mice is therefore not due to the three amino acid substitutions identified within the sequence which is homologous to beta A4 of humans. Moreover, peptides corresponding to residues 1-27 of human and rodent beta A4 are solubilized under physiological conditions; thus they are very unlikely to form stable filaments in vivo.     

pH-dependent structural transitions of Alzheimer amyloid peptides.

To understand the molecular interactions leading to the assembly of beta/44 protein into the hallmark fibrils of Alzheimer's disease (AD), we have examined the ability of synthetic peptides that correspond to the beta/A4 extracellular sequence to form fibrils over the range of pH 3-10. Peptides included the sequences 1-28, 19-28, 17-28, 15-28, 13-28, 11-28, and 9-28 of beta/A4. The model fibrils were compared with isolated amyloid with respect to morphology, conformation, tinctorial properties, and stability under denaturing conditions. Electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and x-ray diffraction revealed that the ionization states of the amino acid sidechains appeared to be a crucial feature in fibril formation. This was reflected by the ability of several peptides to undergo fibril assembly and disassembly as a function of pH. Comparisons between different beta/A4 sequences demonstrated that the fibrillar structure representative of AD amyloid was dependent upon electrostatic interactions, likely involving His-13 and Asp-23, and hydrophobic interactions between uncharged sidechains contained within residues 17-21. The results also indicated an exclusively beta-sheet conformation for the synthetic (and possibly AD fibrils) in contrast to certain other (e.g., systemic) amyloids.     

Design and characterization of peptides with amphiphilic beta-strand structures

To extend our studies on peptides and proteins with amphiphilic secondary structures, a series of peptides designed to form amphiphilic beta-strand structures was designed, synthesized, and characterized by circular dichroism and infrared spectroscopy. Amphiphilic beta-strand conformations may be likely to appear in a variety of surface-active proteins, including apolipoprotein B and fibronectin. In a beta-strand conformation, the synthetic peptides will possess a hydrophobic face composed of valine side chains and a hydrophilic face composed of alternating acidic (glutamic acid) and basic (ornithine or lysine) residues. The peptides studied had a variety of chain lengths (5, 9, and 13 residues), and had the amino groups either free or protected with the trifluoroacetyl group. While the peptides did not possess a high potential for beta-sheet formation based on the Chou Fasman parameters, they possessed significant beta-sheet content, with up to 90% beta-sheet calculated for the 13-residue protected peptide. The driving force for beta-sheet formation is the potential amphiphilicity of this conformation. The beta-strand conformation of the 13-residue deprotected peptide was stable in 50% trifluoroethanol, 6 M guanidine hydrochloride, and octanol. The peptides are strongly self-associating in water, which would reduce the unfavorable contacts of the hydrophobic residues with water. It is clear that small peptides can be designed to form stable beta-strand conformations.     

Inhibitor discovery targeting the intermediate structure of beta-amyloid peptide on the conformational transition pathway: implications in the aggregation mechanism of beta-amyloid peptide

Abeta peptides cleaved from the amyloid precursor protein are the main components of senile plaques in Alzheimer's disease. Abeta peptides adopt a conformation mixture of random coil, beta-sheet, and alpha-helix in solution, which makes it difficult to design inhibitors based on the 3D structures of Abeta peptides. By targeting the C-terminal beta-sheet region of an Abeta intermediate structure extracted from molecular dynamics simulations of Abeta conformational transition, a new inhibitor that abolishes Abeta fibrillation was discovered using virtual screening in conjunction with thioflavin T fluorescence assay and atomic force microscopy determination. Circular dichroism spectroscopy demonstrated that the binding of the inhibitor increased the beta-sheet content of Abeta peptides either by stabilizing the C-terminal beta-sheet conformation or by inducing the intermolecular beta-sheet formation. It was proposed that the inhibitor prevented fibrillation by blocking interstrand hydrogen bond formation of the pleated beta-sheet structure commonly found in amyloid fibrils. The study not only provided a strategy for inhibitor design based on the flexible structures of amyloid peptides but also revealed some clues to understanding the molecular events involved in Abeta aggregation.     

Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs.

The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta 1-28 through beta 1-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta 1-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/ml. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta 1-47 and beta 1-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/ml in the alpha helix-promoting solvent, 1,1,1,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the beta 1-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.     

A general model of the P2 protein of peripheral nervous system myelin based on secondary structure predictions, tertiary folding principles, and experimental observations

The amino acid sequence of the P2 protein of peripheral myelin was analyzed with regard to regions of probable alpha-helix, beta-structure, beta-turn, and unordered conformation by means of several algorithms commonly used to predict secondary structure in proteins. Because of the high beta-sheet content and virtual absence of alpha-helix shown by the circular dichroic spectra of the protein, a bias was introduced into the algorithms to favor the beta-structure over the alpha-helical conformation. In order to define those beta-sheet residues that could lie on the external hydrophilic surface of the protein and those that could lie in its hydrophobic interior, the predicted beta-strands were examined for charged and uncharged amino acids located at alternating positions in the sequence. The sequential beta-strands in the predicted secondary structure were then ordered into beta-sheets and aligned according to generally accepted tertiary folding principles and certain chemical properties peculiar to the P2 protein. The general model of the P2 protein that emerged was a "Greek key" beta-barrel, consisting of eight antiparallel beta-strands with a two-stranded ribbon of antiparallel beta-structure emerging from one end. The model has an uncharged, hydrophobic core and a highly hydrophilic surface. The two Cys residues, which form a disulfide, occur in a loop connecting two adjacent antiparallel strands. Two hydrophilic loops, each containing a cluster of acidic residues and a single Phe, protrude from one end of the molecule. The general model is consistent with many of the properties of the actual protein, including the relatively weak nature of its association with myelin lipids and the positions of amino acid substitutions. Alternative beta-strand orderings yield three specific models having different interstrand connections across the barrel ends.     

Structural homology of lens crystallins. II. Homology expressed by correlation coefficients and hydropathy profiles

Bovine lens alpha A- and alpha B-crystallin polypeptides show extensive sequence homology with each other, but apparently none with beta Bp- and gamma 2-crystallin. Despite only 30% sequence homology, the latter two proteins are assumed to have a strong correspondence in tertiary structure, consisting of four structurally similar folding units of antiparallel beta-sheet. We have tested for internal structural repeats in all crystallins, and structural homology between crystallins, by comparing various physical properties of the amino acid residues, such as bulkiness and propensity to form beta-sheet and beta-turn structure. Two procedures used a combination of five physical parameters to calculate correlation coefficients. The 4-fold structural repeat in gamma 2-crystallin and the internal duplication in beta Bp-crystallin were readily detectable, as was also the strong structural homology between corresponding folding units in beta Bp- and gamma 2-crystallin. However, for alpha-crystallin polypeptides, no conclusive support was obtained for either a four-unit or a six-unit folding, the two models previously considered by us. The third procedure compared smoothened hydropathy plots, representing hydrophilic and hydrophobic regions along the polypeptide sequences. Hydropathy profiles were found to show strong correspondence, particularly between alpha B-crystallin and beta Bp-crystallin. These observations support a similar 4-fold folding pattern for all bovine crystallins. A possible role in subunit interactions of the N-terminal folding unit, which has hydrophobic surface characteristics in both alpha- and beta-crystallin polypeptides, is proposed.     

Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet

A 23-residue peptide termed BH(9-10) was designed based on a beta-hairpin segment of the single-layer beta-sheet region of Borrelia OspA protein. The peptide contains a large number of charged amino acid residues, and it does not follow the amphipathic pattern that is commonly found in natural beta-sheets. In aqueous solution, the peptide was highly soluble and flexible, with a propensity to form a non-native beta-turn. Trifluoroethanol (TFE) stabilized a native-like beta-turn in BH(9-10). TFE also decreased the level of solubility of the peptide, resulting in peptide precipitation. The precipitation process accompanied a conformational conversion to a beta-sheet structure, as judged with circular dichroism spectroscopy. The precipitate was found to be fibrils similar to those associated with human amyloid diseases. The fibrillization kinetics depended on peptide and TFE concentrations, and had a nucleation step followed by an assembly step. The fibrillization was reversible, and the dissociation reaction involved two phases. TFE appears to induce the fibrils by stabilizing a beta-sheet conformation of the peptide that optimally satisfies hydrogen bonding and electrostatic complementarity. This TFE-induced fibrillization is quite unusual, because most amyloidogenic peptides form fibrils in aqueous solution and TFE disrupts these fibrils. Nevertheless, the BH(9-10) fibrils have similar structure to other fibrils, supporting the emerging idea that polypeptides possess an intrinsic ability to form amyloid-like fibrils. The high level of solubility of BH(9-10), the ability to precisely control fibril formation and dissociation, and the high-resolution structure of the same sequence in the beta-hairpin conformation in the OspA protein provide a tractable experimental system for studying the fibril formation mechanism.     

Solution conformations and aggregational properties of synthetic amyloid beta-peptides of Alzheimer's disease. Analysis of circular dichroism spectra.

The A4 or beta-peptide (39 to 43 amino acid residues) is the principal proteinaceous component of amyloid deposits in Alzheimer's disease. Using circular dichroism (c.d.), we have studied the secondary structures and aggregational properties in solution of 4 synthetic amyloid beta-peptides: beta-(1-28), beta-(1-39), beta-(1-42) and beta-(29-42). The natural components of cerebrovascular deposits and extracellular amyloid plaques are beta-(1-39) and beta-(1-42), while beta-(1-28) and beta-(29-42) are unnatural fragments. The beta-(1-28), beta-(1-39) and beta-(1-42) peptides adopt mixtures of beta-sheet, alpha-helix and random coil structures, with the relative proportions of each secondary structure being strongly dependent upon the solution conditions. In aqueous solution, beta-sheet structure is favored for the beta-(1-39) and beta-(1-42) peptides, while in aqueous solution containing trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP), alpha-helical structure is favored for all 3 peptides. The alpha-helical structure unfolds with increasing temperature and is favored at pH 1 to 4 and pH 7 to 10; the beta-sheet conformation is temperature insensitive and is favored at pH 4 to 7. Peptide concentration studies showed that the beta-sheet conformation is oligomeric (intermolecular), whereas the alpha-helical conformation is monomeric (intramolecular). The rate of aggregation to the oligomeric beta-sheet structure (alpha-helix----random coil----beta-sheet) is also dependent upon the solution conditions such as the pH and peptide concentration; maximum beta-sheet formation occurs at pH 5.4. These results suggest that beta-peptide is not an intrinsically insoluble peptide. Thus, solution abnormalities, together with localized high peptide concentrations, which may occur in Alzheimer's disease, may contribute to the formation of amyloid plaques. The hydrophobic beta-(29-42) peptide adopts exclusively an intermolecular beta-sheet conformation in aqueous solution despite changes in temperature or pH. Therefore, this segment may be the first region of the beta-peptide to aggregate and may direct the folding of the complete beta-peptide to produce the beta-pleated sheet structure found in amyloid deposits. Differences between the solution conformations of the beta-(1-39) and beta-(1-42) peptides suggests that the last 3 C-terminal amino acids are crucial to amyloid deposition.     

Molecular modeling of the amyloid-beta-peptide using the homology to a fragment of triosephosphate isomerase that forms amyloid in vitro

The main component of the amyloid senile plaques found in Alzheimer's brain is the amyloid-beta-peptide (A beta), a proteolytic product of a membrane precursor protein. Previous structural studies have found different conformations for the A beta peptide depending on the solvent and pH used. In general, they have suggested an alpha-helix conformation at the N-terminal domain and a beta-sheet conformation for the C-terminal domain. The structure of the complete A beta peptide (residues 1-40) solved by NMR has revealed that only helical structure is present in A beta. However, this result cannot explain the large beta-sheet A beta aggregates known to form amyloid under physiological conditions. Therefore, we investigated the structure of A beta by molecular modeling based on extensive homology using the Smith and Waterman algorithm implemented in the MPsrch program (Blitz server). The results showed a mean value of 23% identity with selected sequences. Since these values do not allow a clear homology to be established with a reference structure in order to perform molecular modeling studies, we searched for detailed homology. A 28% identity with an alpha/beta segment of a triosephosphate isomerase (TIM) from Culex tarralis with an unsolved three-dimensional structure was obtained. Then, multiple sequence alignment was performed considering A beta, TIM from C.tarralis and another five TIM sequences with known three-dimensional structures. We found a TIM segment with secondary structure elements in agreement with previous experimental data for A beta. Moreover, when a synthetic peptide from this TIM segment was studied in vitro, it was able to aggregate and to form amyloid fibrils, as established by Congo red binding and electron microscopy. The A beta model obtained was optimized by molecular dynamics considering ionizable side chains in order to simulate A beta in a neutral pH environment. We report here the structural implications of this study.     

Conformational behavior of ionic self-complementary peptides

Several de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable beta-sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N-terminus and positively charged arginine or lysine residues located toward the C-terminus. This arrangement of charge balances the alpha-helical dipole moment (C --> N), resulting in a strong tendency to form stable alpha-helices as well. Therefore, these peptides can form both stable alpha-helices and beta-sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable beta-sheet and alpha-helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.     

A molecular model of Alzheimer amyloid beta-peptide fibril formation

Polymerization of the amyloid beta (Abeta) peptide into protease-resistant fibrils is a significant step in the pathogenesis of Alzheimer's disease. It has not been possible to obtain detailed structural information about this process with conventional techniques because the peptide has limited solubility and does not form crystals. In this work, we present experimental results leading to a molecular level model for fibril formation. Systematically selected Abeta-fragments containing the Abeta16-20 sequence, previously shown essential for Abeta-Abeta binding, were incubated in a physiological buffer. Electron microscopy revealed that the shortest fibril-forming sequence was Abeta14-23. Substitutions in this decapeptide impaired fibril formation and deletion of the decapeptide from Abeta1-42 inhibited fibril formation completely. All studied peptides that formed fibrils also formed stable dimers and/or tetramers. Molecular modeling of Abeta14-23 oligomers in an antiparallel beta-sheet conformation displayed favorable hydrophobic interactions stabilized by salt bridges between all charged residues. We propose that this decapeptide sequence forms the core of Abeta-fibrils, with the hydrophobic C terminus folding over this core. The identification of this fundamental sequence and the implied molecular model could facilitate the design of potential inhibitors of amyloidogenesis.     

Structural analysis of amyloid beta peptide fragment (25-35) in different microenvironments

Amyloid beta (Abeta) peptides are one of the classes of amphiphilic molecules that on dissolution in aqueous solvents undergo interesting conformational transitions. These conformational changes are known to be associated with their neuronal toxicity. The mechanism of structural transition involved in the monomeric Abeta to toxic assemblage is yet to be understood at the molecular level. Early results indicate that oriented molecular crowding has a profound effect on their assemblage formation. In this work, we have studied how different microenvironments affect the conformational transitions of one of the active amyloid beta-peptide fragments (Abeta(25-35)). Spectroscopic techniques such as CD and Fourier transform infrared spectroscopy were used. It was observed that a stored peptide concentrates on dissolution in methanol adopts a minor alpha-helical conformation along with unordered structures. On changing the methanol concentration in the solvated film form, the conformation switches to the antiparallel beta-sheet structure on the hydrophilic surface, whereas the peptide shows transition from a mixture of helix and unordered structure into predominantly a beta-sheet with minor contribution of helix structure on the hydrophobic surface. Our present investigations indicate that the conformations induced by the different surfaces dictate the gross conformational preference of the peptide concentrate.     

Solution structure of the Alzheimer amyloid beta-peptide (1-42) in an apolar microenvironment. Similarity with a virus fusion domain.

The major components of neuritic plaques found in Alzheimer disease (AD) are peptides known as amyloid beta-peptides (Abeta), which derive from the proteolitic cleavage of the amyloid precursor proteins. In vitro Abeta may undergo a conformational transition from a soluble form to aggregated, fibrillary beta-sheet structures, which seem to be neurotoxic. Alternatively, it has been suggested that an alpha-helical form can be involved in a process of membrane poration, which would then trigger cellular death. Conformational studies on these peptides in aqueous solution are complicated by their tendency to aggregate, and only recently NMR structures of Abeta-(1-40) and Abeta-(1-42) have been determined in aqueous trifluoroethanol or in SDS micelles. All these studies hint to the presence of two helical regions, connected through a flexible kink, but it proved difficult to determine the length and position of the helical stretches with accuracy and, most of all, to ascertain whether the kink region has a preferred conformation. In the search for a medium which could allow a more accurate structure determination, we performed an exhaustive solvent scan that showed a high propensity of Abeta-(1-42) to adopt helical conformations in aqueous solutions of fluorinated alcohols. The 3D NMR structure of Abeta-(1-42) shows two helical regions encompassing residues 8-25 and 28-38, connected by a regular type I beta-turn. The surprising similarity of this structure, as well as the sequence of the C-terminal moiety, with those of the fusion domain of influenza hemagglutinin suggests a direct mechanism of neurotoxicity.     

Conformation-activity relationship of peptide T and new pseudocyclic hexapeptide analogs.

Peptide T (ASTTTNYT), a segment corresponding to residues 185-192 of gp120, the coat protein of HIV, has several important biological properties in vitro that have stimulated the search for simpler and possibly more active analogs. We have previously shown that pseudocyclic hexapeptide analogs containing the central residues of peptide T retain considerable chemotactic activity. We have now extended the design of this type of analogs to peptides containing different aromatic residues and/or Ser in lieu of Thr. The complex conformation-activity relationship of these analogs called for a reexamination of the basic conformational tendencies of peptide T itself. Here, we present an exhaustive NMR conformational study of peptide T in different media. Peptide T assumes a gamma-turn in aqueous mixtures of ethylene glycol, a type-IV beta-turn conformation in aqueous mixtures of DMF, and a type-II beta-turn conformation in aqueous mixtures of DMSO. The preferred conformations for the analogs were derived from modeling, starting from the preferred conformations of peptide T. The best models derived from the gamma-turn conformation of peptide T are those of peptides XII (DSNYSR), XIII (ETNYTK) and XVI (ESNYSR). The best models derived from the type-IV beta-turn conformation of peptide T are those of peptides XIV (KTTNYE) and XV (DSSNYR). No low-energy models could be derived starting from the type-II beta-turn conformation of peptide T. The analogs with the most favored conformations are also the most active in the chemotactic test.     

Analysis of the secondary structure of beta-amyloid (Abeta42) fibrils by systematic proline replacement

Amyloid fibrils in Alzheimer's disease mainly consist of 40- and 42-mer beta-amyloid peptides (Abeta40 and Abeta42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of Abeta peptides are rich in intermolecular beta-sheet, the precise secondary structure of Abeta in the aggregates remains unclear. To identify the amino acid residues involved in the beta-sheet formation, 34 proline-substituted mutants of Abeta42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in beta-sheet, whereas they are easily accommodated in beta-turn as a Pro-X corner. Among the mutants at positions 15-32, only E22P-Abeta42 extensively aggregated with stronger neurotoxicity than wild-type Abeta42, suggesting that the residues at positions 15-21 and 24-32 are involved in the beta-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of Abeta42. The C-terminal proline mutants (A42P-, I41P-, and V40P-Abeta42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T- and I41T-Abeta42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of Abeta42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the beta-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between Abeta42 and Abeta40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type Abeta42. The identification of the beta-sheet region of Abeta42 is a basis for designing new aggregation inhibitors of Abeta peptides.     

Spectroscopic evidence for amyloid-like interfacial self-assembly of hydrophobin Sc3

Amphipathic fungal proteins called hydrophobins are able to self-assemble into insoluble supramolecular structures at hydrophobic/hydrophilic interfaces, but the molecular mechanism and underlying protein conformation changes are not known. Secondary-structure prediction indicated that hydrophobin Sc3 is an all-beta protein. Many amyloidogenic proteins self-assemble into insoluble amyloid fibrils while undergoing a change to an all-beta conformation. In this study we show that two dyes, thioflavin T, and Congo red, which are widely used for specific detection of stacked beta sheets, interact with Sc3 assemblies in the same way as with the amyloid beta-sheet fibrils. We conclude that Sc3, and probably other hydrophobins too, self-assemble at interfaces in the same manner as amyloidogenic proteins, i.e., through beta-sheet stacking.     

Self-assembling peptides: sequence, secondary structure in solution and film formation

Peptides of alternating charge and hydrophobic amino acids have a tendency to adopt unusually stable beta-sheet structures that can form insoluble macroscopic aggregates under physiological conditions. In this study, analogues of a well-known self-assembling peptide, characterized by the same polar/nonpolar periodicity but with different residues, were designed to study the relationship between sequence, conformation in solution and film-forming capacity in saline solution. Peptide conformation, evaluated by circular dichroism, correlated with film forming capacity observed by inverted optical microscopy after addition of saline solution and subsequent drying. We found that polar/nonpolar periodicity of several analogues is not criterion enough to induce beta-sheet and thus film formation and that conformations different from beta-sheet also allow self-assemblage. Furthermore, addition of the short adhesive sequence RGD to a known self-assembling sequence was shown to not prevent the self-assembling process. This finding might prove useful for the design of biomimetic scaffolds. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 906-915, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.