Helix Stabilization Precedes Aqueous and Bilayer-Catalyzed Fiber Formation in Islet Amyloid Polypeptide

Islet amyloid polypeptide (IAPP) is an unstructured polypeptide hormone that is cosecreted with insulin. In patients with type 2 diabetes, IAPP undergoes a transition from its natively disordered state to a highly ordered, all-β-strand amyloid fiber. Although predominantly disordered, IAPP transiently samples α-helical structure in solution. IAPP adopts a fully helical structure when bound to membrane surfaces in a process associated with catalysis of amyloid formation. Here, we use spectroscopic techniques to study the structure of full-length, monomeric IAPP under amyloidogenic conditions. We observe that the residues with helical propensity in solution (1-22) also form the membrane-associated helix. Additionally, reduction of the N-terminal disulfide bond (Cys2-Cys7) decreases the extent of helix formed throughout this region. Through manipulation of sample conditions to increase or decrease the amount of helix, we show that the degree of helix formed affects the rate of amyloid assembly. Formation of helical structure is directly correlated with enhanced amyloid formation both on the membrane surface and in solution. These observations support suggested mechanisms in which parallel helix associations bring together regions of the peptide that could nucleate β-strand structure. Remarkably, stabilization of non-amyloid structure appears to be a key intermediate in assembly of IAPP amyloid.     

A peptide study of the relationship between the collagen triple-helix and amyloid

Type XXV collagen, or collagen‐like amyloidogenic component, is a component of amyloid plaques, and recent studies suggest this collagen affects amyloid fibril elongation and has a genetic association with Alzheimer's disease. The relationship between the collagen triple helix and amyloid fibrils was investigated by studying peptide models, including a very stable triple helical peptide (Pro‐Hyp‐Gly)₁₀, an amyloidogenic peptide GNNQQNY, and a hybrid peptide where the GNNQQNY sequence was incorporated between (GPO)_n domains. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy showed the GNNQQNY peptide formed a random coil structure, whereas the hybrid peptide contained a central disordered GNNQQNY region transitioning to triple‐helical ends. Light scattering confirmed the GNNQQNY peptide had a high propensity to form amyloid fibrils, whereas amyloidogenesis was delayed in the hybrid peptide. NMR data suggested the triple‐helix constraints on the GNNQQNY sequence within the hybrid peptide may disfavor the conformational change necessary for aggregation. Independent addition of a triple‐helical peptide to the GNNQQNY peptide under aggregating conditions delayed nucleation and amyloid fibril growth. The inhibition of amyloid nucleation depended on the Gly‐Xaa‐Yaa sequence and required the triple‐helix conformation. The inhibitory effect of the collagen triple‐helix on an amyloidogenic sequence, when in the same molecule or when added separately, suggests Type XXV collagen, and possibly other collagens, may play a role in regulating amyloid fibril formation. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 795–806, 2012.     

Stabilization of helical structure in two 17-residue amphipathic analogues of the C-terminal peptide of cytochrome C

The conformations of two 17-residue peptide analogues derived from the C-terminal sequence of pigeon cytochrome c (native sequence = KAERADLIAYLKQATAK) were examined in aqueous and lipid environments by CD spectroscopy. The two analogues, KKLLKKLIAYLKQATAK (K peptide) and EELLEELIAYLKQATAK (E peptide), were made amphipathic with respect to helical segregation by substituting a 6-residue sequence at the N-terminus of the native peptide. Their structures were compared to the native peptide under aqueous conditions of varying pH and temperature, and in the presence of liposomes composed of phosphatidylcholine and phosphatidylserine in the ratio of 9:1. The results indicated that the native peptide remains unstructured under all the conditions examined even though this region of the native molecule is surface exposed and helical. The E peptide, however, was helical under aqueous conditions at 25 degrees C from pH 2-10 with a maximum helicity at pH 4 (54% helix from analysis of CD data). The ellipticity of the E peptide at pH 4 and 8 was concentration dependent, indicating an aggregation phenomenon. In studies in which the CD spectrum was measured at different temperatures, the E peptide became more helical at lower temperatures at pH 4 but not at pH 8. Upon interaction with a lipid membrane in the form of liposomes, there appeared to be a slight destabilization in the structure of the E peptide. The K peptide in an aqueous environment behaved like the native peptide in that it was structureless at all pHs and temperatures examined. In the presence of liposomes, however, this peptide had a high helical content (75% helix from analysis of CD data). These findings suggest that while stabilization of the helix dipole with negative charges at the N-terminus are important in inducing helical conformation in the E peptide, hydrophobic interactions created during aggregation appear to provide the principal stabilizing force. The results with the K peptide demonstrate that the positive N-terminal sequence of this peptide is able to interact with the negatively charged head groups in the phospholipid membrane in such a fashion as to stabilize a helical structure that is not apparent in an aqueous environment alone.     

Role of the helical structure of the N-terminal region of Plasmodium falciparum merozoite surface protein 2 in fibril formation and membrane interaction

Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP2(1-25)) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP2(1-25) in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP2(1-25) was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP2(1-25) was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP2(1-25) in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10-24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP2(1-25) fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.     

Zinc boosts EGCG's hIAPP amyloid Inhibition both in solution and membrane

Amyloid aggregation of human islet amyloid polypeptide (hIAPP) is linked to insulin-producing islet cell death in type II diabetes. Previous studies have shown that zinc (Zn(II)) and insulin, co-secreted with hIAPP, have an inhibition effect on hIAPP aggregation. Lipid membranes have also been shown to significantly influence the aggregation kinetics of hIAPP. An increasing number of studies report the importance of developing small molecule inhibitors to suppress the hIAPP's aggregation and subsequent toxicity. The ability of epigallocatechin-gallate (EGCG) to inhibit aggregation of a variety of amyloid peptide/proteins initiated numerous studies as well as the development of derivative compounds to potentially treat amyloid diseases. In this study, a combination of Thioflavin-T fluorescence kinetics, transmission electron microscopy, isothermal titration calorimetery, circular dicrosim and nucelar magnetic resonance experiments were used to demonstrate a significant enhancement in EGCG's efficiency when complexed with Zn(II). We demonstrate that the Zn-EGCG complex is able to significantly suppress hIAPP's amyloid aggregation both in presence and absence of lipid membrane. Circular dichroism experiments indicate the formation and stabilization of a helical structure of hIAPP in presence of the EGCG:Zn(II) complex. Our results also reveal the ability of EGCG or EGCG:Zn(II) to efficiently suppress hIAPP's cellular toxicity. We believe that the reported results could be useful to develop strategies to trap hIAPP intermediates for further biophysical and structural studies, and also to devise approaches to abolish amyloid aggregation and cellular toxicity.     

NMR solution structure and membrane interaction of the N-terminal sequence (1-30) of the bovine prion protein

The structure and membrane interaction of the N-terminal sequence (1-30) of the bovine prion protein (bPrPp) has been investigated by NMR spectroscopy in phospholipid membrane mimetic systems. CD spectroscopy revealed that the peptide adopts a largely alpha-helical structure in zwitterionic bicelles as well as in DHPC micelles but has a less degree of alpha-helix structure in partly charged bicelles. The solution structure of bPrPp was determined in DHPC micelles, and an alpha-helix was found between residues Ser8 and Ile21. The residues within the helical region show slow amide hydrogen exchange. Translational diffusion measurements in zwitterionic q = 0.5 bicelles show that the peptide does not induce aggregation of the bicelles. Increased quadrupolar splittings were observed in the outer part of the (2)H spectrum of DMPC in q = 3.5 bicelles, indicating that the peptide induces a certain degree of order in the bilayer. The amide hydrogen exchange and the (2)H NMR results are consistent with a slight positive hydrophobic mismatch and that bPrPp forms a stable helix that inserts in a transmembrane location in the bilayer. The structure of bPrPp and its position in the membrane may be relevant for the understanding of how the N-terminal (1-30) part of the bovine PrP functions as a cell-penetrating peptide. These findings may lead to a better understanding of how the prion protein accumulates at the membrane surface and also how the conversion into the scrapie form is carried out.     

Folding and membrane insertion of amyloid‐beta (25–35) peptide and its mutants: Implications for aggregation and neurotoxicity

The mechanisms of interfacial folding and membrane insertion of the Alzheimer's amyloid‐β fragment Aβ(25–35) and its less toxic mutant, N27A‐Aβ(25–35) and more toxic mutant, M35A‐Aβ(25–35), are investigated using replica–exchange molecular dynamics in an implicit water‐membrane environment. This study simulates the processes of interfacial folding and membrane insertion in a spontaneous fashion to identify their general mechanisms. Aβ(25–35) and N27A‐Aβ(25–35) peptides share similar mechanisms: the peptides are first located in the membrane hydrophilic region where their C‐terminal residues form helical structures. The peptides attempt to insert themselves into the membrane hydrophobic region using the C‐terminal or central hydrophobic residues. A small portion of peptides can successfully enter the membrane's hydrophobic core, led by their C‐terminal residues, through the formation of continuous helical structures. No detectable amount of M35A‐Aβ(25–35) peptides appeared to enter the membrane's hydrophobic core. The three studied peptides share a similar helical structure for their C‐terminal five residues, and these residues mainly buried within the membrane's hydrophobic region. In contrast, their N‐terminal properties are markedly different. With respect to the Aβ(25–35), the N27A‐Aβ(25–35) forms a more structured helix and is buried deeper within the membrane, which may result in a lower degree of aggregation and a lower neurotoxicity; in contrast, the less structured and more water‐exposed M35A‐Aβ(25–35) is prone to aggregation and has a higher neurotoxicity. Understanding the mechanisms of Aβ peptide interfacial folding and membrane insertion will provide new insights into the mechanisms of neurodegradation and may give structure‐based clues for rational drug design preventing amyloid associated diseases. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Conversion of non-fibrillar beta-sheet oligomers into amyloid fibrils in Alzheimer's disease amyloid peptide aggregation

Abeta(1-40) is one of the main components of the fibrils found in amyloid plaques, a hallmark of brains affected by Alzheimer's disease. It is known that prior to the formation of amyloid fibrils in which the peptide adopts a well-ordered intermolecular beta-sheet structure, peptide monomers associate forming low and high molecular weight oligomers. These oligomers have been previously described in electron microscopy, AFM, and exclusion chromatography studies. Their specific secondary structures however, have not yet been well established. A major problem when comparing aggregation and secondary structure determinations in concentration-dependent processes such as amyloid aggregation is the different concentration range required in each type of experiment. In the present study we used the dye Thioflavin T (ThT), Fourier-transform infrared spectroscopy, and electron microscopy in order to structurally characterize the different aggregated species which form during the Abeta(1-40) fibril formation process. A unique sample containing 90microM peptide was used. The results show that oligomeric species which form during the lag phase of the aggregation kinetics are a mixture of unordered, helical, and intermolecular non-fibrillar beta-structures. The number of oligomers and the amount of non-fibrillar beta-structures grows throughout the lag phase and during the elongation phase these non-fibrillar beta-structures are transformed into fibrillar (amyloid) beta-structures, formed by association of high molecular weight intermediates.     

Solution structure of human growth hormone-releasing factor fragment (1-29) by CD: characteristic conformational change on phospholipid membrane

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution. In addition, although the peptide exists as almost complete helix in the 50 vol% aqueous alcohol (80-90% helicity), it does not reach full helicity even in the solution containing excess amount of phospholipid liposome (maximum 65-70% helicity). The conformational difference is explained by the characteristic amphipathy of the peptide, i.e., the necessity to twist the separated amphipathic helical parts in the interaction with the phospholipid membrane probably makes the helicity of the peptide decrease.     

Aggregation-induced conformation changes dictate islet amyloid polypeptide (IAPP) membrane affinity

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates (“oligomers”) of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic “oligomer fold” may not be universal.     

Study of the conformational transition of A beta(1-42) using D-amino acid replacement analogues

A critical event in Alzheimer's disease is the transition of Abeta peptides from their soluble forms into disease-associated beta-sheet-rich conformers. Structural analysis of a complete D-amino acid replacement set of Abeta(1-42) enabled us to localize in the full-length 42-mer peptide the region responsible for the conformational switch into a beta-sheet structure. Although NMR spectroscopy of trifluoroethanol-stabilized monomeric Abeta(1-42) delineated two separated helical domains, only the destabilization of helix I, comprising residues 11-24, caused a transition to a beta-sheet structure. This conformational alpha-to-beta switch was directly accompanied by an aggregation process leading to the formation of amyloid fibrils.     

Secondary structure of substance P bound to liposomes in organic solvents and in solution from Raman and CD spectroscopy.

The membrane lipid phase may be an important mediator of the peptide-receptor interaction. In order to understand the mechanism of this interaction, it is important to know the peptide structure, not only in the hydrophobic lipid bilayer environment, but also at the bilayer surface and in solution. To investigate this problem we have measured the secondary structure of the 11-residue neuropeptide substance P (SP) and its fragments in aqueous solutions, in membrane mimetic solvents, and associated with lipid bilayers using Raman and CD spectroscopy. Raman and CD spectra of SP bound to liposomes indicate a less than 20% helix content. We interpret these results to indicate that SP contains virtually no helix when bound to negatively charged liposomes. These spectra are similar to spectra of peptides in type I and III beta-turns. SP forms between 10 and 30% (1-3 residues) helical structure in sodium dodecyl sulfate micelles and less than 10% helix in methanol and trifluoroethanol. The binding of SP to negatively charged liposomes significantly changes the structure of the lipid acyl chains, decreasing order in some cases and increasing it in others. Raman spectra of SP in water indicates that SP near 30 mM forms an ensemble of structures in water that is distinct from completely unfolded peptide and from the aggregated beta-sheet form observed in saline solutions. We conclude from our CD results that methods used to quantitate secondary structure from CD spectra of short peptides cannot be used to distinguish between very short helical segments and beta-turns.     

Structural analysis and amphiphilic properties of a chemically synthesized mitochondrial signal peptide

Anionic phospholipids induce a marked conformational change in a synthetic peptide corresponding to residues 1-27 of pre-ornithine carbamyltransferase. The peptide designated, pO-(1-27)-peptide amide, becomes more alpha-helical in the presence of cardiolipin or dimyristoylphosphatidylglycerol but not in the presence of dimyristoylphosphatidylcholine. The greater helix-promoting action of anionic versus zwitterionic lipids is predicted by helix-coil transition theory. This statistical mechanical theory also predicts that a shorter peptide, N-acetyl-pO-(16-27)-peptide amide, has less helix-forming tendency, even in the presence of sodium dodecyl sulfate, despite the fact that it has a comparable number of positive charges. The N-acetyl-pO-(16-27)-peptide amide has no helical structure in buffer with or without dimyristoylphosphatidylglycerol but it has a small (5%) helical content in methanol. Thus, the ability of anionic lipids to promote helix formation requires more than the presence of cationic groups on the peptide. The angular dependence of the hydrophobic moment of the putative helical segment of pO-(1-27)-peptide amide demonstrates that any helical structure which is formed would have some amphiphilic character. The pO-(1-27)-peptide amide disrupts large lipid aggregates to form discoid micelles about 30 to 50 nm in diameter. The ability to lyse membranes into disc-shaped micelles is characteristic of peptides containing an amphiphathic helix. In the case of the mitochondrial signal peptide, this membrane-lytic behavior may contribute to the translocation of the protein into the organelle.     

Conformational polymorphism of the amyloidogenic peptide homologous to residues 113-127 of the prion protein

Conformational transitions are thought to be the prime mechanism of amyloid formation in prion diseases. The prion proteins are known to exhibit polymorphic behavior that explains their ability of "conformation switching" facilitated by structured "seeds" consisting of transformed proteins. Oligopeptides containing prion sequences showing the polymorphism are not known even though amyloid formation is observed in these fragments. In this work, we have observed polymorphism in a 15-residue peptide PrP (113-127) that is known to form amyloid fibrils on aging. To see the polymorphic behavior of this peptide in different solvent environments, circular dichroism (CD) spectroscopic studies on an aqueous solution of PrP (113-127) in different trifluoroethanol (TFE) concentrations were carried out. The results show that PrP (113-127) have sheet preference in lower TFE concentration whereas it has more helical conformation in higher TFE content (>40%). The structural transitions involved in TFE solvent were studied using interval-scan CD and FT-IR studies. It is interesting to note that the alpha-helical structure persists throughout the structural transition process involved in amyloid fibril formation implicating the involvement of both N- and C-terminal sequences. To unravel the role of the N-terminal region in the polymorphism of the PrP (113-127), CD studies on another synthetic peptide, PrP (113-120) were carried out. PrP(113-120) exhibits random coil conformation in 100% water and helical conformation in 100% TFE, indicating the importance of full-length sequence for beta-sheet formation. Besides, the influence of different chemico-physical conditions such as concentration, pH, ionic strength, and membrane like environment on the secondary structure of the peptide PrP (113-127) has been investigated. At higher concentration, PrP (113-127) shows features of sheet conformation even in 100% TFE suggesting aggregation. In the presence of 5% solution of sodium dodecyl sulfate, PrP (113-127) takes high alpha-helical propensity. The environment-dependent conformational polymorphism of PrP (113-127) and its marked tendency to form stable beta-sheet structure at acidic pH could account for its conformation switching behavior from alpha-helix to beta-sheet. This work emphasizes the coordinative involvement of N-terminal and C-terminal sequences in the self-assembly of PrP (113-127).     

Structures of the transmembrane helices of the G-protein coupled receptor, rhodopsin.

An hypothesis is tested that individual peptides corresponding to the transmembrane helices of the membrane protein, rhodopsin, would form helices in solution similar to those in the native protein. Peptides containing the sequences of helices 1, 4 and 5 of rhodopsin were synthesized. Two peptides, with overlapping sequences at their termini, were synthesized to cover each of the helices. The peptides from helix 1 and helix 4 were helical throughout most of their length. The N- and C-termini of all the peptides were disordered and proline caused opening of the helical structure in both helix 1 and helix 4. The peptides from helix 5 were helical in the middle segment of each peptide, with larger disordered regions in the N- and C-termini than for helices 1 and 4. These observations show that there is a strong helical propensity in the amino acid sequences corresponding to the transmembrane domain of this G-protein coupled receptor. In the case of the peptides from helix 4, it was possible to superimpose the structures of the overlapping sequences to produce a construct covering the whole of the sequence of helix 4 of rhodopsin. As similar superposition for the peptides from helix 1 also produced a construct, but somewhat less successfully because of the disordering in the region of sequence overlap. This latter problem was more severe for helix 5 and therefore a single peptide was synthesized for the entire sequence of this helix, and its structure determined. It proved to be helical throughout. Comparison of all these structures with the recent crystal structure of rhodopsin revealed that the peptide structures mimicked the structures seen in the whole protein. Thus similar studies of peptides may provide useful information on the secondary structure of other transmembrane proteins built around helical bundles.     

Characterization of Aβ aggregation mechanism probed by congo red

β-Amyloid peptide (1) (Aβ) aggregates are toxic to neuron and the main cause of Alzheimer's disease (AD). The role of congo red (CR) on Aβ aggregation is controversial in aqueous solution. Both prevention and promotion of Aβ aggregation have been proposed, suggesting that CR may interact with Aβ of different structural conformations resulting in different effects on Aβ aggregation behavior. CR with these characteristics can be applied to probe the molecular mechanism of Aβ aggregation. Therefore, in the present study, we used CR as a probe to study the Aβ aggregation behavior in sodium dodecyl sulfate (SDS) condition. Our results show that Aβ(40) adopts two short helices at Q15-S26 and K28-L34 in the SDS environment. CR can interact with the helical form of Aβ(40), and the main interaction site is located at the first helical and hydrophobic core region, residues 17-25, which is assigned as a discordant helix region. Furthermore, CR may prevent Aβ(40) undergoing α-helix to β-strand conversion, and therefore aggregation through stabilizing the helical conformation of discordant helix in SDS environment, suggesting that the discordant helix plays a key role on the conformational stabilization of Aβ. Our present study implies that any factors or molecules that can stabilize the discordant helical conformation may also prevent the Aβ aggregation in membrane associated state. This leads to a new therapeutic strategy for the development of lead compounds to AD.     

Total synthesis of the natural,medium-length,peptaibol pentadecaibin and study of the chemical features responsible for its membrane activity

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, C^α-tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol–membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure–activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes.     

Conformational switching and fibrillogenesis in the amyloidogenic fragment of apolipoprotein a-I

The N-terminal portion of apolipoprotein A-I corresponding to the first 93 residues has been identified as the main component of apolipoprotein A-I fibrils in a form of systemic amyloidosis. We have been able to characterize the process of conformational switching and fibrillogenesis in this fragment of apolipoprotein A-I purified directly from ex vivo amyloid material. The peptide exists in an unstructured form in aqueous solution at neutral pH. The acidification of the solution provokes a collapse into a more compact, intermediate state and the transient appearance of a helical conformation that rapidly converts to a stable, mainly beta-structure in the fibrils. The transition from helical to sheet structure occurs concomitantly with peptide self-aggregation, and fibrils are detected after 72 h. The alpha-helical conformation is induced by the addition of trifluoroethanol and phospholipids. Interaction of the amyloidogenic polypeptide with phospholipids prevents the switching from helical to beta-sheet form and inhibits fibril formation. The secondary structure propensity of the apolipoprotein A-I fragment appears poised between helix and the beta-sheet. These findings reinforce the idea of a delicate balance between natively stabilizing interactions and fatally stabilizing interactions and stress the importance of cellular localization and environment in the maintenance of protein conformation.     

A single mutation in the nonamyloidogenic region of islet amyloid polypeptide greatly reduces toxicity

Islet amyloid polypeptide (IAPP or amylin) is a 37-residue peptide secreted with insulin by beta-cells in the islets of Langerhans. The aggregation of the peptide into either amyloid fibers or small soluble oligomers has been implicated in the death of beta-cells during type 2 diabetes through disruption of the cellular membrane. The actual form of the peptide responsible for beta-cell death has been a subject of controversy. Previous research has indicated that the N-terminal region of the peptide (residues 1-19) is primarily responsible for the membrane-disrupting effect of the hIAPP peptide and induces membrane disruption to a similar extent as the full-length peptide without forming amyloid fibers when bound to the membrane. The rat version of the peptide, which is both noncytotoxic and nonamyloidogenic, differs from the human peptide by only one amino acid residue: Arg18 in the rat version while His18 in the human version. To elucidate the effect of this difference, we have measured in this study the effects of the rat and human versions of IAPP(1-19) on islet cells and model membranes. Fluorescence microscopy shows a rapid increase in intracellular calcium levels of islet cells after the addition of hIAPP(1-19), indicating disruption of the cellular membrane, while the rat version of the IAPP(1-19) peptide is significantly less effective. Circular dichroism experiments and dye leakage assays on model liposomes show that rIAPP(1-19) is deficient in binding to and disrupting lipid membranes at low but not at high peptide to lipid ratios, indicating that the ability of rIAPP(1-19) to form small aggregates necessary for membrane binding and disruption is significantly less than hIAPP(1-19). At pH 6.0, where H18 is likely to be protonated, hIAPP(1-19) resembles rIAPP(1-19) in its ability to cause membrane disruption. Differential scanning calorimetry suggests a different mode of binding to the membrane for rIAPP(1-19) compared to hIAPP(1-19). Human IAPP(1-19) has a minimal effect on the phase transition of lipid vesicles, suggesting a membrane orientation of the peptide in which the mobility of the acyl chains of the membrane is relatively unaffected. Rat IAPP(1-19), however, has a strong effect on the phase transition of lipid vesicles at low concentrations, suggesting that the peptide does not easily insert into the membrane after binding to the surface. Our results indicate that the modulation of the peptide orientation in the membrane by His18 plays a key role in the toxicity of nonamyloidogenic forms of hIAPP.     

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMRsolution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide Abeta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native Abeta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length Abeta peptides Abeta(1-40) and Abeta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which Abeta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of Abeta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation.