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.     

Conserved and cooperative assembly of membrane-bound alpha-helical states of islet amyloid polypeptide

The conversion of soluble protein into beta-sheet-rich amyloid fibers is the hallmark of a number of serious diseases. Precursors for many of these systems (e.g., Abeta from Alzheimer's disease) reside in close association with a biological membrane. Membrane bilayers are reported to accelerate the rate of amyloid assembly. Furthermore, membrane permeabilization by amyloidogenic peptides can lead to toxicity. Given the beta-sheet-rich nature of mature amyloid, it is seemingly paradoxical that many precursors are either intrinsically alpha-helical or transiently adopt an alpha-helical state upon association with membrane. In this work, we investigate these phenomena in islet amyloid polypeptide (IAPP). IAPP is a 37-residue peptide hormone which forms amyloid fibers in individuals with type II diabetes. Fiber formation by human IAPP (hIAPP) is markedly accelerated by lipid bilayers despite adopting an alpha-helical state on the membrane. We further show that IAPP partitions into monomeric and oligomeric helical assemblies. Importantly, it is this latter state which most strongly correlates to both membrane leakage and accelerated fiber formation. A sequence variant of IAPP from rodents (rIAPP) does not form fibers and is reputed not to permeabilize membranes. Here, we report that rIAPP is capable of permeabilizing membranes under conditions that permit rIAPP membrane binding. Sequence and spectroscopic comparisons of rIAPP and hIAPP enable us to propose a general mechanism for the helical acceleration of amyloid formation in vitro. As rIAPP cannot form amyloid fibers, our results show that fiber formation need not be directly coupled to toxicity.     

All-atom structure ensembles of islet amyloid polypeptides determined by enhanced sampling and experiment data restraints

Exploring the accurate structure ensembles are critical to understand the functions of intrinsically disordered proteins (IDPs). As a well-known IDP, islet amyloid polypeptide (IAPP) plays important roles in the development of human type II diabetes (T2D). The toxicity of human IAPP (hIAPP) is induced by the amyloidosis of the peptide, however, its aggregation mechanism remains ambiguous. The characterization of structure ensemble of hIAPP, as well as the differences between hIAPP and its non-amyloidogenic homologous such as rat IAPP (rIAPP), would greatly help to illuminate the amyloidosis mechanism of IAPP. In this study, the atomic structure ensembles of hIAPP and rIAPP were characterized by all-atom molecular dynamics (MD) simulations combined with enhanced sampling technology and experiment data restraints. The obtained structure ensembles were firstly compared with those determined by the conventional MD (cMD) and enhanced sampling without experiment data restraints. The results showed that the enhanced sampling and experiment data restraints would improve the simulation accuracy. The transient N-terminal α-helix structures were adopted by the sub-states of both hIAPP and rIAPP, however, the C-terminal helical structures were only present on hIAPP. The hydrophobic residues in the amyloid-core region of hIAPP are exposed to the solvent. The structure ensemble differences between hIAPP and rIAPP revealed in this work provide potential explain to the amyloidogenic mechanism and would be helpful for the design of drugs to combat T2D.     

Induction of endoplasmic reticulum stress-induced beta-cell apoptosis and accumulation of polyubiquitinated proteins by human islet amyloid polypeptide

The islet in type 2 diabetes is characterized by an approximately 60% beta-cell deficit, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). Human IAPP (hIAPP) but not rodent IAPP (rIAPP) forms toxic oligomers and amyloid fibrils in an aqueous environment. We previously reported that overexpression of hIAPP in transgenic rats triggered endoplasmic reticulum (ER) stress-induced apoptosis in beta-cells. In the present study, we sought to establish whether the cytotoxic effects of hIAPP depend on its propensity to oligomerize, rather than as a consequence of protein overexpression. To accomplish this, we established a novel homozygous mouse model overexpressing rIAPP at a comparable expression rate and, on the same background, as a homozygous transgenic hIAPP mouse model previously reported to develop diabetes associated with beta-cell loss. We report that by 10 wk of age hIAPP mice develop diabetes with a deficit in beta-cell mass due to increased beta-cell apoptosis. The rIAPP transgenic mice counterparts do not develop diabetes or have decreased beta-cell mass. Both rIAPP and hIAPP transgenic mice have increased expression of BiP, but only hIAPP transgenic mice have elevated ER stress markers (X-box-binding protein-1, nuclear localized CCAAT/enhancer binding-protein homologous protein, active caspase-12, and accumulation of ubiquitinated proteins). These findings indicate that the beta-cell toxic effects of hIAPP depend on the propensity of IAPP to aggregate, but not on the consequence of protein overexpression.     

Comparing the structural properties of human and rat islet amyloid polypeptide by MD computer simulations

Conformational properties of the full-length human and rat islet amyloid polypeptide 1-37 (amyloidogenic hIAPP and non-amyloidogenic rIAPP, respectively) were studied at 310 and 330 K by MD simulations both for the cysteine (reduced IAPP) and cystine (oxidized IAPP) moieties. At all temperatures studied, IAPP does not adopt a well-defined conformation and is essentially random coil in solution, although transient helices appear forming along the peptide between residues 8 and 22, particularly in the reduced form. Above the water percolation transition (at 320 K), the reduced hIAPP moiety presents a considerably diminished helical content remaining unstructured, while the natural cystine moiety reaches a rather compact state, presenting a radius of gyration that is almost 10% smaller and characterized by intrapeptide H-bonds that form many β-bridges in the C-terminal region. This compact conformation presents a short end-to-end distance and seems to form through the formation of β-sheet conformations in the C-terminal region with a minimization of the Y/F distances in a two-step mechanism: the first step taking place when the Y37/F23 distance is ~ 1.1 nm, and subsequently Y37/F15 reaches its minimum of ~ 0.86 nm. rIAPP, which does not aggregate, also presents transient helical conformations. A particularly stable helix is located in proximity of the C-terminal region, starting from residues L27 and P28. Our MD simulations show that P28 in rIAPP influences the secondary structure of IAPP by stabilizing the peptide in helical conformations. When this helix is not present, the peptide presents bends or H-bonded turns at P28 that seem to inhibit the formation of the β-bridges seen in hIAPP. Conversely, hIAPP is highly disordered in the C-terminal region, presenting transient isolated β-strand conformations, particularly at higher temperatures and when the natural disulfide bond is present. Such conformational differences found in our simulations could be responsible for the different aggregational propensities of the two different homologues. In fact, the fragment 30-37, which is identical in both homologues, is known to aggregate in vitro, hence the overall sequence must be responsible for the amyloidogenicity of hIAPP. The increased helicity in rIAPP induced by the serine-to-proline variation at residue 28 seems to be a plausible inhibitor of its aggregation.     

A single mutation on the human amyloid polypeptide modulates fibril growth and affects the mechanism of amyloid-induced membrane damage

Amyloid fibril formation has been implicated in a wide range of human diseases and the interactions of amyloidogenic proteins with cell membranes are considered to be important in the aetiology of these pathologies. In type 2 diabetes mellitus (T2DM), the human islet amyloid polypeptide (hIAPP) forms amyloid fibrils which impair the functionality and viability of pancreatic β cells. The mechanisms of hIAPP cytotoxicity are linked to the ability of the peptide to self-aggregate and to interact with membranes. Previous studies have shown that the N-terminal part of hIAPP from residues 1 to 19 is the membrane binding domain. The non-amyloidogenic and nontoxic mouse IAPP differs from hIAPP by six residues out of 37, among which a single one, residue 18, lies in the membrane binding region. To gain more insight into hIAPP-membrane interactions we herein performed comprehensive biophysical studies on four analogues (H18R-IAPP, H18K-IAPP, H18E-IAPP and H18A-IAPP). Our data reveal that all peptides are able to insert efficiently in the membrane, indicating that residue 18 is not essential for hIAPP membrane binding and insertion. However, only wild-type hIAPP and H18K-IAPP are able to form fibrils at the membrane. Importantly, all peptides induce membrane damage; wild-type hIAPP and H18K-IAPP presumably cause membrane disruption mainly by fibril growth at the membrane, while for H18R-IAPP, H18E-IAPP and H18A-IAPP, membrane leakage is most likely due to high molecular weight oligomeric species. These results highlight the importance of the residue at position 18 in IAPP for modulating fibril formation at the membrane and the mechanisms of membrane leakage.     

Sequence and Crowding Effects in the Aggregation of a 10-Residue Fragment Derived from Islet Amyloid Polypeptide

Fibril formation from amyloidogenic peptides is a hallmark of a wide range of diseases, including Alzheimer's disease and type II diabetes. Characterization of the aggregation process should include intrinsic factors, such as sequence variation, and extrinsic factors, such as crowding effects. To this end, we examined the interactions of dimers composed of residues 20-29 of human islet amyloid polypeptide (hIAPP), which form fibrils in vitro, and the nonamyloidogenic rat IAPP (rIAPP) using molecular dynamics simulations modeled at different peptide concentrations. There is a substantial free energy barrier to unbind the hIAPP dimer whereas no barrier exists for separating the rIAPP dimer. The profound differences in the free energy landscapes of the rIAPP and hIAPP dimers explains the lack of fibril formation in hIAPP upon substitution of the C-terminal residues by proline. Enhancing the extent of crowding has a substantial effect on both the barrier for separating a hIAPP β-sheet dimer and the formation of potential β-sheet nucleation sites. Our results show that the propensity for forming nucleation sites is dependent not only on the amino-acid sequence but also on the context in which it is found.     

Islet amyloid polypeptide (IAPP) competes for two binding sites of CGRP.

Two distinct binding sites for [125I]human calcitonin gene-related peptide (hCGRP) were found in rat brain, skeletal muscle, and liver. Each tissue had a high affinity site with an average Kd of 46 pM and a low affinity site with an average Kd of 22 nM. Islet amyloid polypeptide (IAPP), which has N- and C-terminal sequence homology to CGRP and is produced by islet beta-cells, bound to both sites but had a potency closer to that of CGRP at the low affinity binding site. A C-terminal fragment of IAPP competed for [125I]hCGRP binding at the low affinity site with potency comparable to that of hIAPP. No specific binding to membrane preparations was found in experiments using [125I]rIAPP, which was iodinated at the C-terminal tyrosyl residue. These results suggest that some of the previously reported biological effects occurring at nM or microM concentrations of IAPP may be mediated by IAPP binding to low affinity CGRP receptors. This study further indicates that the C-terminal region of IAPP is important for binding to low affinity CGRP receptors, and suggests that C-terminal fragments of IAPP may be of biological importance.     

Liposome Damage and Modeling of Fragments of Human Islet Amyloid Polypeptide (IAPP) Support a Two-Step Model of Membrane Destruction

A key factor in the development of Type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide (hIAPP) is believed to play a crucial role in this process by forming small aggregates that disrupt the cellular membrane. During Type II diabetes mellitus, human IAPP (hIAPP) fibrillizes to form amyloid deposits. However, the role of various regions of the 37 amino acid peptide in the process of membrane disruption has yet to be fully elucidated. Therefore, several fragments (10–19, 20–29, 10–29, 1–19) of hIAPP were synthesized and compared to full length hIAPP for their effects on model PC/PS bilayers. These fragments were also modeled using density functional methods and analyzed by circular dichroism, to determine possible correlations between activity and available conformations and charge distribution. Results from dye leakage and Thioflavin T fluorescence assays confirmed that the hIAPP fragments disrupt the lipid bilayer to varying extents, despite their inability to form amyloid fibrils. The longer and more positively charged fragments were most active in the assay (1–19 > 10–29 > 10–19 > 20–29), though none rivaled the activity of the native full length peptide. This may reflect their relative abilities to interact with the negatively charged membrane. Data support a two-step model for membrane disruption: insertion by the N-terminus followed by fibrillization mediated by the middle to C-terminal region.     

Conformational preferences of the amylin nucleation site in SDS micelles: an NMR study

Human islet amyloid polypeptide (hIAPP), or amylin, is a 37 amino acid hormone secreted by pancreatic beta-cells. hIAPP constitutes approximately 90% of the amyloid deposits found in type II diabetic patients. It has been shown that the central region of the peptide (hIAPP(20-29)) constitutes the nucleation site for the amyloidogenic process with F23 playing a key role in the formation of the beta-pleated structures. In addition, it has been proposed that an important stage in the cytotoxicity of hIAPP is its interaction with the beta-cell membranes. As a first step toward the characterization of the interaction of hIAPP with cell membranes, we determined conformational preferences of hIAPP(20-29) in membrane-mimicking environments. We found that upon interacting with negatively charged micelles, the dominant conformation of hIAPP(20-29) is a distorted type I beta-turn centered on residues F23 and G24, with F23, A25, and I26 forming a small hydrophobic cluster that may facilitate the interaction of this peptide with the membrane bilayer. Moreover, we were able to elucidate the topological orientation of the peptide that is absorbed on the micelle surface, with the hydrophobic cluster oriented toward the hydrocarbon region of the micelles and both N- and C-termini exposed to the solvent.     

Structure and membrane orientation of IAPP in its natively amidated form at physiological pH in a membrane environment

Human islet amyloid polypeptide is a hormone coexpressed with insulin by pancreatic beta-cells. For reasons not clearly understood, hIAPP aggregates in type II diabetics to form oligomers that interfere with beta-cell function, eventually leading to the loss of insulin production. The cellular membrane catalyzes the formation of amyloid deposits and is a target of amyloid toxicity through disruption of the membrane's structural integrity. Therefore, there is considerable current interest in solving the 3D structure of this peptide in a membrane environment. NMR experiments could not be directly utilized in lipid bilayers due to the rapid aggregation of the peptide. To overcome this difficulty, we have solved the structure of the naturally occurring peptide in detergent micelles at a neutral pH. The structure has an overall kinked helix motif, with residues 7-17 and 21-28 in a helical conformation, and with a 3(10) helix from Gly 33-Asn 35. In addition, the angle between the N- and C-terminal helices is constrained to 85°. The greater helical content of human IAPP in the amidated versus free acid form is likely to play a role in its aggregation and membrane disruptive activity.     

Cloning and expression of human islet amyloid polypeptide in cultured cells

Efforts to clone amyloidogenic proteins in the cells often have resulted in cell death. We report successful cloning and expression of recombinant human islet amyloid polypeptide (hIAPP) in cultured mammalian cells. Amylin gets secreted, forms fibrils that are toxic to target cells like beta cells of rat and human. The study involves cloning of full-length amylin in fluorescent protein vector followed by transfection into mammalian cells. The transfected cells with recombinant human amylin, secrete the translated protein corresponding to 37-amino acid native mature IAPP. The mature IAPP secreted out of the cell is purified and characterized by MALDI-TOF/TOF-MS and Western blotting. Purified IAPP forms fibrils as seen by Thioflavin-T fluorescence and AFM, and these fibrils were cytotoxic towards pancreatic cell line RIN5mf cells.     

Identification of a novel human islet amyloid polypeptide beta-sheet domain and factors influencing fibrillogenesis

Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent beta-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to beta-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state.     

Identification of minimal peptide sequences in the (8-20) domain of human islet amyloid polypeptide involved in fibrillogenesis

We have examined a series of overlapping peptide fragments from the 8-20 region of human islet amyloid polypeptide (IAPP) with the objective of defining the smallest fibril-forming domain. Peptide fragments corresponding to LANFLV (residues 12-17) and FLVHSS (residues 15-20) were strong enhancers of beta-sheet transition and fibril formation. Negative stain electron microscopy illustrated the ability of these peptide fragments to form fibrils independently when incubated alone in solution. Circular dichroism analysis revealed that when full-length human IAPP was incubated in the presence of these two fragments, fibrillogenesis was accelerated. While the two fragments, LANFLV and FLVHSS, were able to enhance the recruitment of additional IAPP molecules during fibril formation, the "seeding" activity of these peptides had no effect on altering IAPP-induced cytotoxcity as determined by cell culture studies. Therefore, this study has identified two internal IAPP peptide fragments within the 8-20 domain that may have a role in enhancing the folding and aggregation of human IAPP. These fragments are the smallest sequences identified, within the 8-20 region of hIAPP, that can independently form fibrils, and that can interact with IAPP to assemble into fibrils with characteristics similar as those formed by human IAPP alone.     

NMR solution structure of the peptide fragment 1-30, derived from unprocessed mouse Doppel protein, in DHPC micelles

The downstream prion-like Doppel (Dpl) protein is a homologue related to the prion protein (PrP). Dpl is expressed in the brains of mice that do not express PrP, and Dpl is known to be toxic to neurons. One mode of toxicity has been suggested to involve direct membrane interactions. PrP under certain conditions of cell trafficking retains an uncleaved signal peptide, which may also hold for the much less studied Dpl. For a peptide with a sequence derived from the N-terminal part (1-30) of mouse Dpl (mDpl(1-30)) CD spectroscopy shows about 40% alpha-helical structure in DHPC and SDS micelles. In aqueous solution it is mostly a random coil. The three-dimensional solution structure was determined by NMR for mDpl(1-30) associated with DHPC micelles. 2D 1H NMR spectra of the peptide in q = 0.25 DMPC/DHPC bicelles only showed signals from the unstructured termini, indicating that the structured part of the peptide resides within the lipid bilayer. Together with 2H2O exchange data in the DHPC micelle solvent, these results show an alpha-helix protected from solvent exchange between residues 7 and 19, and suggest that the alpha-helical segment can adopt a transmembrane localization also in a membrane. Leakage studies with entrapped calcein in large unilamellar phospholipid vesicles showed that the peptide is almost as membrane perturbing as melittin, known to form pores in membranes. The results suggest a possible channel formation mechanism for the unprocessed Dpl protein, which may be related to toxicity through direct cell membrane interaction and damage.     

Amyloidogenicity of naturally occurring full‐length animal IAPP variants

The aggregation of the 37‐amino acid polypeptide human islet amyloid polypeptide (hIAPP), as either insoluble amyloid or as small oligomers, appears to play a direct role in the death of human pancreatic β‐islet cells in type 2 diabetes. hIAPP is considered to be one of the most amyloidogenic proteins known. The quick aggregation of hIAPP leads to the formation of toxic species, such as oligomers and fibers, that damage mammalian cells (both human and rat pancreatic cells). Whether this toxicity is necessary for the progression of type 2 diabetes or merely a side effect of the disease remains unclear. If hIAPP aggregation into toxic amyloid is on‐path for developing type 2 diabetes in humans, islet amyloid polypeptide (IAPP) aggregation would likely need to play a similar role within other organisms known to develop the disease. In this work, we compared the aggregation potential and cellular toxicity of full‐length IAPP from several diabetic and nondiabetic organisms whose aggregation propensities had not yet been determined for full‐length IAPP.     

Destabilization of human IAPP amyloid fibrils by proline mutations outside of the putative amyloidogenic domain: is there a critical amyloidogenic domain in human IAPP?

Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.     

Neutron diffraction reveals sequence-specific membrane insertion of pre-fibrillar islet amyloid polypeptide and inhibition by rifampicin

Human islet amyloid polypeptide (hIAPP) forms amyloid deposits in non-insulin-dependent diabetes mellitus (NIDDM). Pre-fibrillar hIAPP oligomers (in contrast to monomeric IAPP or mature fibrils) increase membrane permeability, suggesting an important role in the disease. In the first structural study of membrane-associated hIAPP, lamellar neutron diffraction shows that oligomeric hIAPP inserts into phospholipid bilayers, and extends across the membrane. Rifampicin, which inhibits hIAPP-induced membrane permeabilisation in functional studies, prevents membrane insertion. In contrast, rat IAPP (84% identical to hIAPP, but non-amyloidogenic) does not insert into bilayers. Our findings are consistent with the hypothesis that membrane-active pre-fibrillar hIAPP oligomers insert into beta cell membranes in NIDDM.     

Comparison of the conformation and electrostatic surface properties of magainin peptides bound to sodium dodecyl sulfate and dodecylphosphocholine micelles

The role played by noncovalent interactions in inducing a stable secondary structure onto the sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelle-bound conformations of (Ala(8,13,18))magainin 2 amide and the DPC micelle bound conformation of magainin 1 were determined. Two-dimensional NMR and molecular modeling investigations indicated that (Ala(8,13,18))magainin 2 amide bound to DPC micelles adopts a alpha-helical secondary structure involving residues 2-16. The four C-terminal residues converge to a lose beta-turn structure. (Ala(8,13,18))magainin 2 amide bound to SDS miscelles adopts a alpha-helical secondary structure involving residues 7-18. The C- and N-terminal residues exhibited a great deal of conformational flexibility. Magainin 1 bound to DPC micelles adopts a alpha-helical secondary structure involving residues 4-19. The C-terminal residues converge to a lose beta-turn structure. The results of this investigation indicate hydrophobic interactions are the major contributors to stabilizing the induced helical structure of the micelle-bound peptides. Electrostatic interactions between the polar head groups of the micelle and the cationic side chains of the peptides define the positions along the peptide backbone where the helical structures begin and end.     

Structural characteristics of the beta-sheet-like human and rat islet amyloid polypeptides as determined by scanning tunneling microscopy

We demonstrate in this work that scanning tunneling microscopy (STM) provides a useful approach to obtaining structural information about human islet amyloid polypeptide (hIAPP) and rat islet amyloid polypeptide (rIAPP) assembly on highly oriented pyrolytic graphite (HOPG) with sub-molecular resolution. The observed hIAPP and rIAPP lamellae consisted of parallel stripes. The STM images of hIAPPs show multiple molecular folding structures, with an average of 11 amino acid residues for the core regions. In addition, the STM images also reveal the assembly characteristics of rIAPP lamellae and may indicate a secondary structural conformation from random coil to beta-sheet-like on the graphite surface.