Comparison of molecular dynamics simulation methods for amyloid β(1-42) monomers containing D-aspartic acid residues for predicting retention times in chromatography

Molecular dynamics simulations of amyloid β(1-42) containing D-aspartic acid residues were performed using several continuous solvent models to investigate the usefulness of simulation methods for D-amino acid-containing proteins and peptides. Normal molecular dynamics simulations and replica exchange molecular dynamics simulations, which are one of the generalized-ensemble algorithms, were performed. Because the β-structure contents of amyloid β(1-42) peptides obtained by replica exchange molecular dynamics simulations with Onufriev-Bashford-Case generalized Born implicit solvent were qualitatively consistent with experimental data, replica exchange molecular dynamics rather than other methods appeared to be more reasonable for calculations of amyloid β(1-42) containing D-aspartic acid residues. Computational results revealed that peptides with stereoinversion of Asp23 tend to form β-sheet structures by themselves, in contrast to the wild-type peptides that form β-sheet structures only after aggregation. These results are expected to be useful for computational investigations of proteins and peptides such as prediction of retention time of peptides and proteins containing D-aspartic acid residues.     

Limitations of Peptide Retro-inverso Isomerization in Molecular Mimicry

A retro-inverso peptide is made up of d-amino acids in a reversed sequence and, when extended, assumes a side chain topology similar to that of its parent molecule but with inverted amide peptide bonds. Despite their limited success as antigenic mimicry, retro-inverso isomers generally fail to emulate the protein-binding activities of their parent peptides of an α-helical nature. In studying the interaction between the tumor suppressor protein p53 and its negative regulator MDM2, Sakurai et al. (Sakurai, K., Chung, H. S., and Kahne, D. (2004) J. Am. Chem. Soc. 126, 16288–16289) made a surprising finding that the retro-inverso isomer of p53(15–29) retained the same binding activity as the wild type peptide as determined by inhibition enzyme-linked immunosorbent assay. The authors attributed the unusual outcome to the ability of the d-peptide to adopt a right-handed helical conformation upon MDM2 binding. Using a battery of biochemical and biophysical tools, we found that retro-inverso isomerization diminished p53 (15,–29) binding to MDM2 or MDMX by 3.2–3.3 kcal/mol. Similar results were replicated with the C-terminal domain of HIV-1 capsid protein (3.0 kcal/mol) and the Src homology 3 domain of Abl tyrosine kinase (3.4 kcal/mol). CD and NMR spectroscopic as well as x-ray crystallographic studies showed that d-peptide ligands of MDM2 invariably adopted left-handed helical conformations in both free and bound states. Our findings reinforce that the retro-inverso strategy works poorly in molecular mimicry of biologically active helical peptides, due to inherent differences at the secondary and tertiary structure levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structure level.     

Folding simulations of Trp‐cage mini protein in explicit solvent using biasing potential replica‐exchange molecular dynamics simulations

Replica exchange molecular dynamics (RexMD) simulations are frequently used for studying structure formation and dynamics of peptides and proteins. A significant drawback of standard temperature RexMD is, however, the rapid increase of the replica number with increasing system size to cover a desired temperature range. A recently developed Hamiltonian RexMD method has been used to study folding of the Trp‐cage protein. It employs a biasing potential that lowers the backbone dihedral barriers and promotes peptide backbone transitions along the replica coordinate. In two independent applications of the biasing potential RexMD method including explicit solvent and starting from a completely unfolded structure the formation of near‐native conformations was observed after 30–40 ns simulation time. The conformation representing the most populated cluster at the final simulation stage had a backbone root mean square deviation of ～1.3 Å from the experimental structure. This was achieved with a very modest number of five replicas making it well suited for peptide and protein folding and refinement studies including explicit solvent. In contrast, during five independent continuous 70 ns molecular dynamics simulations formation of collapsed states but no near native structure formation was observed. The simulations predict a largely collapsed state with a significant helical propensity for the helical domain of the Trp‐cage protein already in the unfolded state. Hydrogen bonded bridging water molecules were identified that could play an active role by stabilizing the arrangement of the helical domain with respect to the rest of the chain already in intermediate states of the protein. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Peptide folding simulations

Developments in the design of small peptides that mimic proteins in complexity, recent advances in nanosecond time-resolved spectroscopy methods to study peptides and the development of modern, highly parallel simulation algorithms have come together to give us a detailed picture of peptide folding dynamics. Two newly implemented simulation techniques, parallel replica dynamics and replica exchange molecular dynamics, can now describe directly from simulations the kinetics and thermodynamics of peptide formation, respectively. Given these developments, the simulation community now has the tools to verify and validate simulation protocols and models (forcefields).     

Retro-inverso peptide analogues of Trypanosoma cruzi B13 protein epitopes fail to be recognized by human sera and peripheral blood mononuclear cells

Retro inverso (RI) analogues of antigenic synthetic peptides, which are made of D-amino acids with a reversed sequence, may mimic the side chain conformation of natural all-L peptides. RI analogues were cross-reactively recognized by antibodies and CD4+ T cells reactive against natural all-L synthetic peptides or native proteins in animal models. Since peptides containing D-amino acids are highly resistant to proteolytic digestion, cross-reactive RI analogues may be ideal for in vivo administration to humans as synthetic peptide vaccines or immunomodulators. B13 is an immunodominant tandemly repetitive protein from Trypanosoma cruzi, a protozoan parasite that is the causative antigen of Chagas' disease. In order to test whether RI peptides can be recognized by human antibody and T cells, we synthesized two all-L peptides containing the immunodominant B (S12) and T (S15.7) cell epitopes of B13 protein from T. cruzi and their retro (R, made of all-L amino acids with reversed sequence), inverso (I, made of all-D amino acids) and RI analogues. Recognition of peptides S12, S12-R, S12-I and S12-RI by anti-B13 antibodies in sera from T. cruzi-infected patients was tested in competitive ELISA assay with recombinant B13 protein as the solid phase antigen. Peptides S15.7 and its topological analogues were tested at the 10-50 microM range in proliferation assays on peripheral blood mononuclear cells (PBMC) from S15.7-responder individuals. The median percentage inhibition of B13 ELISA for peptide S12 was 94%, while those of the RI analogue or the other topological analogues were below 12%. While peptide S15.7 was recognized by PBMC from all subjects tested, none recognized the RI analogue of the S15.7 T cell epitope. Our results indicate that cross-reactivity with natural epitopes is not an universal property of RI analogues. This may limit the general applicability of the use of cross-reactive RI analogues as human vaccines and immunotherapeutic agents.     

Antimicrobial activity and stability to proteolysis of small linear cationic peptides with D-amino acid substitutions

Antimicrobial peptides contribute to innate host defense against a number of bacteria and fungal pathogens. Some of antimicrobial synthetic peptides were systemically administered in vivo; however, effective protection has so far not been obtained because the effective dose of peptides in vivo seems to be very high, often close to the toxic level against the host. Alternatively, peptides administered in vivo may be degraded by certain proteases present in serum. In this study, D-amino acids were substituted for the L-amino acids of antimicrobial peptides to circumvent these problems. Initially a peptide (L-peptide) rich in five arginine residues and consisting of an 11-amino acid peptide (residues 32-42) of human granulysin was synthesized. Subsequently, the L-amino acids of the 11-amino acid peptide were replaced partially (D-peptide) or wholly (AD-peptide) with D-amino acids. Activity and stability to proteolysis, in particular, in the serum of antimicrobial peptides with D-amino acid substitutions were examined. Peptides with D-amino acid substitutions were found to lyse bacteria as efficiently as their all-L-amino acid parent, L-peptide. In addition, the peptide composed of L-amino acids was susceptible to trypsin, whereas peptides containing D-amino acid substitutions were highly stable to trypsin treatment. Similarly, the peptide consisting of L-amino acids alone was also susceptible to fetal calf serum (FCS), however, protease inhibitors restored the lowered antimicrobial activity of the FCS-incubated peptide. Thus, D-amino acid substitutions can make antimicrobial peptides resistant to proteolysis, suggesting that the antimicrobial peptides consisting of D-amino acids are potential candidates for clinical therapeutic use.     

Structure of Met-enkephalin in explicit aqueous solution using replica exchange molecular dynamics.

Replica exchange molecular dynamics (MD) simulations of Met-enkephalin in explicit solvent reveal helical and nonhelical structures. Four predominant structures of Met-enkephalin are sampled with comparable probabilities (two helical and two nonhelical). The energy barriers between these configurations are low, suggesting that Met-enkephalin switches easily between configurations. This is consistent with the requirement that Met-enkephalin be sufficiently flexible to bind to several different receptors. Replica exchange simulations of 32 ns are shown to sample approximately five times more configurational space than constant temperature MD simulations of the same duration. The energy landscape for the replica exchange simulation is presented. A detailed study of replica trajectories demonstrates that the significant increases in temperature provided by the replica exchange technique enable transitions from nonhelical to helical structures that would otherwise be prevented by kinetic trapping. Met-enkephalin (Type Entrez Proteins; Value A61445; Service Entrez Proteins).     

Reproducible polypeptide folding and structure prediction using molecular dynamics simulations

The folding of a polypeptide from an extended state to a well-defined conformation is studied using microsecond classical molecular dynamics (MD) simulations and replica exchange molecular dynamics (REMD) simulations in explicit solvent and in vacuo. It is shown that the solvated peptide folds many times in the REMD simulations but only a few times in the conventional simulations. From the folding events in the classical simulations we estimate an approximate folding time of 1-2 micros. The REMD simulations allow enough sampling to deduce a detailed Gibbs free energy landscape in three dimensions. The global minimum of the energy landscape corresponds to the native state of the peptide as determined previously by nuclear magnetic resonance (NMR) experiments. Starting from an extended state it takes about 50 ns before the native structure appears in the REMD simulations, about an order of magnitude faster than conventional MD. The calculated melting curve is in good qualitative agreement with experiment. In vacuo, the peptide collapses rapidly to a conformation that is substantially different from the native state in solvent.     

Enhanced sampling of peptide and protein conformations using replica exchange simulations with a peptide backbone biasing-potential

During replica exchange molecular dynamics (RexMD) simulations, several replicas of a system are simulated at different temperatures in parallel allowing for exchange between replicas at frequent intervals. This technique allows significantly improved sampling of conformational space and is increasingly being used for structure prediction of peptides and proteins. A drawback of the standard temperature RexMD is the rapid increase of the replica number with increasing system size to cover a desired temperature range. In an effort to limit the number of replicas, a new Hamiltonian-RexMD method has been developed that is specifically designed to enhance the sampling of peptide and protein conformations by applying various levels of a backbone biasing potential for each replica run. The biasing potential lowers the barrier for backbone dihedral transitions and promotes enhanced peptide backbone transitions along the replica coordinate. The application on several peptide cases including in all cases explicit solvent indicates significantly improved conformational sampling when compared with standard MD simulations. This was achieved with a very modest number of 5-7 replicas for each simulation system making it ideally suited for peptide and protein folding simulations as well as refinement of protein model structures in the presence of explicit solvent.     

Cyclic retro-inverso dipeptides with two aromatic side chains. I. Synthesis.

A series of cyclic retro-inverso dipeptides--2-[(4-hydroxy)benzyl]-5-benzyl-4,6(1H,2H,3H,5H)-pyrimidinedi one (c[mPhe-gTyr]), 2-benzyl-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[mTyr-gPhe]), and 2-benzyl-5-amino-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[(alpha-amino)mTyr-gPhe])--were synthesized in order to define the minimum structural requirements for binding affinity with opiate receptors and biological activity. Although the first two compounds lack a free amine proposed to be necessary for receptor recognition, the c[mPhe-gTyr] and c[mTyr-gPhe] analogues serve as model molecules in conformational studies of the target analogue, c[(alpha-amino)mTyr-gPhe]. The cis- and trans-c[(alpha-amino)mTyr-gPhe] contain all the functional groups such as the amine and phenolic groups in the tyrosine, and the aromatic group in the phenylalanine, necessary for opiate activity. In addition, the c[(alpha-amino)mTyr-gPhe] analogues possess similar geometries to the Tyr-Pro part of morphiceptin (Tyr-Pro-Phe-Pro-NH2) whose high mu-receptor activity is attributed to conformations with the Tyr-Pro amide bond in a cis conformation because the peptide bonds assume a cis conformation. However, both analogues are inactive in the guinea pig ileum and the mouse vas deferens assays. This may result from wrong orientation of the benzyl group of the gPhe residue with respect to the (alpha-amino)mTyr residue. Conformational studies of these molecules using 1H-nmr spectroscopy and molecular mechanics calculations will be reported in the following paper. Results of conformational analysis should provide information about backbone-side-chain interactions in the retro-inverso peptide chains since all the fundamental structural elements of the retro-inverso peptides are included in these model systems even though the peptide bonds must assume a cis conformation.     

The investigation of the secondary structure propensities and free-energy landscapes of peptide ligands by replica exchange molecular dynamics simulations

The conformational states of two peptide sequences that bind to staphylococcal enterotoxin B are sampled by replica exchange molecular dynamic (REMD) simulations in explicit water. REMD simulations were treated with 52 replicas in the range of 280–501 K for both peptides. The conformational ensembles of both peptides are dominated by random coil, bend and turn structures with a small amount of helical structures for each temperature. In addition, while an insignificant presence of β-bridge structures were observed for both peptides, the β-sheet structure was observed only for peptide 3. The results obtained from simulations at 300 K are consistent with the experimental results obtained from circular dichroism spectroscopy. From the analysis of REMD results, we also calculated hydrophobic and hydrophilic solvent accessible surface areas for both peptides, and it was observed that the hydrophobic segments of the peptides tend to form bend or turn structures. Moreover, the free-energy landscapes of both peptides were obtained by principal component analysis to understand how the secondary structural properties change according to their complex space. From the free-energy analysis, we have found several minima for both peptides at decreased temperature. For these obvious minima of both peptides, it was observed that the random coil, bend and turn structures are still dominant and the helix, β-bridge or β-sheet structures can appear or disappear with respect to minima. On the other hand, when we compare the results of REMD with conventional MD simulations for these peptides, the configurations of peptide 3 might be trapped in energy minima during the conventional MD simulations. Hence, it can be said that the REMD simulations have provided a sufficiently high sampling efficiency.     

Why the OPLS-AA force field cannot produce the β-hairpin structure of H1 peptide in solution when comparing with the GROMOS 43A1 force field?

The optimal combination of force field and water model is an essential problem that is able to increase molecular dynamics simulation quality for different types of proteins and peptides. In this work, an attempt has been made to explore the problem by studying H1 peptide using four different models based on different force fields, water models and electrostatic schemes. The driving force for H1 peptide conformation transition and the reason why the OPLS-AA force field cannot produce the β-hairpin structure of H1 peptide in solution while the GROMOS 43A1 force field can do were investigated by temperature replica exchange molecular dynamics simulation (T-REMD). The simulation using the GROMOS 43A1 force field preferred to adopt a β-hairpin structure, which was in good agreement with the several other simulations and the experimental evidences. However, the simulation using the OPLS-AA force field has a significant difference from the simulations with the GROMOS 43A1 force field simulation. The results show that the driving force in H1 peptide conformation transition is solvent exposure of its hydrophobic residues. However, the subtle balances between residue-residue interactions and residue-solvent interaction are disrupted by using the OPLS-AA force field, which induced the reduction in the number of residue-residue contact. Similar solvent exposure of the hydrophobic residues is observed for all the conformations sampled using the OPLS-AA force field. For H1 peptide which exhibits large solvent exposure of the hydrophobic residues, the GROMOS 43A1 force field with the SPC water model can provide more accurate results.     

Structural comparison between retro-inverso and parent peptides: Molecular basis for the biological activity of a retro-inverso analogue of the immunodominant fragment of VP1 coat protein from foot-and-mouth disease virus

Antibodies induced against intact foot-and-mouth disease Virus (FMDV) particles bind to the retro-inverso analogue of fragment 141–159 of the viral coat protein VP1 of FMDV, variant A, equally well as to the parent peptide. A conformational investigation of this retro-inverso peptide was carried out by nmr spectroscopy and restrained molecular modeling in order to identify the structural basis for the antigenic mimicry between these retro-inverso and parent peptides. In 100% trifluoroethanol a well-defined left-handed α-helical region exists from residue 150 to residue 159, which is consistently present in all conformational families obtained from restrained modelling. A less-defined left-handed helical region is present in the tract 144–148, which is also consistent for all structures. Conformational flexibility exists about Gly149, which leads to two types of structures, either bent or linear. In the bent structures, a three-residue inverse tight turn is found, which can be classified as an inverse γ-turn centered at Gly149. The overall structural features of the retro-inverso peptide are shown to be similar to those of the parent L-peptide. The two molecules, however, are roughly mirror images because they share inherently chiral secondary structure elements. By comparing these conformational conclusions with the x-ray structure of the Fab complex of a corresponding VP1 antigenic fragment, a rationale is proposed to account for the topological requirements of specific recognition that are implied by the equivalent antigenic activity of the natural and retro-inverso compounds. © 1997 John Wiley & Sons, Inc. Biopoly 41: 569–590, 1997.     

Lactam constraints provide insights into the receptor-bound conformation of secretin and stabilize a receptor antagonist

The natural ligands for family B G protein-coupled receptors are moderate-length linear peptides having diffuse pharmacophores. The amino-terminal regions of these ligands are critical for biological activity, with their amino-terminal truncation leading to production of orthosteric antagonists. The carboxyl-terminal regions of these peptides are thought to occupy a ligand-binding cleft within the disulfide-bonded amino-terminal domains of these receptors, with the peptides in amphipathic helical conformations. In this work, we have characterized the binding and activity of a series of 11 truncated and lactam-constrained secretin(5-27) analogues at the prototypic member of this family, the secretin receptor. One peptide in this series with lactam connecting residues 16 and 20 [c[E(16),K(20)][Y(10)]sec(5-27)] improved the binding affinity of its unconstrained parental peptide 22-fold while retaining the absence of endogenous biological activity and competitive antagonist characteristics. Homology modeling with molecular mechanics and molecular dynamics simulations established that this constrained peptide occupies the ligand-binding cleft in an orientation similar to that of natural full-length secretin and provided insights into why this peptide was more effective than other truncated conformationally constrained peptides in the series. This lactam bridge is believed to stabilize an extended α-helical conformation of this peptide while in solution and not to interfere with critical residue-residue approximations while docked to the receptor.     

Molecular modeling of conformational properties of oligodepsipeptides

A depsipeptide is a chemical structure consisting of both ester and amide bonds. Quantum mechanics calculations have been performed to investigate the conformational properties of a depsidipeptide in the gas and solution phases. Similar to an alanine dipeptide, the depsidipeptide exhibits a strong preference for the polyproline II (PPII) helical conformation. Meanwhile, due to the changes in the intramolecular interaction, the propensity for beta-sheets and alpha-helices diminishes while an unusual inclination for the (phi,psi) = (-150 degrees ,0 degrees ) conformation was observed. A molecular mechanics model has been developed for polydepsipeptides based on the quantum mechanical study. Both simulated annealing and replica exchange molecular dynamics simulations have been carried out on oligodepsipeptide sequences with alternating depsi and natural residues in solution. Novel helical structures have been indicated from the simulations. When glycine is used as the alternating natural amino acid residue, the PPII conformation of a depsi residue stabilizes the peptide into a right-handed helical structure while the alpha-helical conformation of the depsi residue favors an overall left-handed helical structure. The free energy analysis indicates that both the left- and the right-handed helices are equally likely to exist. When charged lysine is introduced as the alternating natural residue, however, it is found that the depsipeptide sequence prefers an extended conformation as in PPII. Our results indicate that the depsipeptide is potentially useful in designing protein mimetics with controllable structure, function, and chemistry.     

Synthesis and anti-aggregatory activity of linear retro-inverso RGD peptides.

Six retro-inverso tri- and tetrapeptide analogues of RGD were prepared and their anti-aggregatory activity was determined by platelet aggregation tests in comparison with the corresponding parent peptides. An efficient method for the introduction of a malonyl-aspartic residue into a peptide chain is described for the first time. A 2-3-fold decrease in potency or total loss of bioactivity was observed with the new peptides; structure-activity relationships are discussed.     

An inhibitory peptide derived from the α-subunit of the epithelial sodium channel (ENaC) shows a helical conformation

Proteolytic activation of the heteromeric epithelial sodium channel (ENaC) is thought to involve the release of inhibitory peptides from the extracellular domains of its α- and γ-subunit. Recently, we demonstrated that an α-13-mer peptide, corresponding to a putative inhibitory region within the extracellular domain of human αENaC, inhibits human αβγENaC. The aim of the present study was to investigate the structural basis of the inhibitory effect of this α-13-mer peptide. Analysis of the peptide by replica exchange molecular dynamics method, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations suggested that a helical turn at the carboxy-terminus is the preferred conformational state of the α-13-mer peptide. From this we predicted that a specific mutation (leucine 188 to alanine) should have a strong effect on the conformational preferences of the peptide. To functionally test this, we compared the effect of the wild-type α-13-mer with that of a mutant α-L188A-13-mer on ENaC currents in Xenopus laevis oocytes heterologously expressing human αβγENaC. We demonstrated that replacing the leucine 188 by alanine abolished the inhibitory effect of the α-13-mer peptide on ENaC. These findings suggest that a helical conformation in its carboxyterminal part is functionally important to mediate ENaC inhibition by the α-13-mer peptide. However, high resolution structural information on the complex of the inhibitory αENaC peptide and the channel are needed to confirm this conclusion.     

Modeling of peptides connecting the ligand‐binding and transmembrane domains in the GluR2 glutamate receptor

Ligand‐gated Glutamate receptors (GluR) mediate synaptic signals in the nervous system. Ionotropic GluRs of AMPA type, the subject of this study, are tetrameric assemblies of monomer subunits, each of which is constructed in a modular fashion from functional subdomains. The extracellular ligand‐binding domain (LBD) changes its conformation upon binding of an agonist ligand followed by opening of a transmembrane (TM) ion channel. Peptides connecting the LBD and TM domains facilitate gating of the channel, and their structure and composition are important for the receptor functioning. In this study, we used replica exchange molecular dynamics (REMD) simulations to model S1M1 and S2M3 connecting peptides of the GluR2 receptor in two implicit solvents, water and interfacial water/lipid medium characterized by lower polarity. Propensity of these peptides to form helical structures was analyzed using helicity measure derived from the free energy of the simulated ensembles of structures. The S1M1 and S2M3 connecting peptides were not helical in our simulations in both dielectric environments in the absence of the rest of the protein. The structures of the LBD fragment with known high‐resolution α‐helical structure and of the TM3 helix were successfully predicted in the simulations, which in part validate our results. The S2M3 peptide, which is important in gating, formed a well‐defined coil structure and salt‐bridges with the S2 domain. The S1M1 peptide formed a loop structure via formation of internal salt‐bridges. Potential implications of these structures on function of the receptor are discussed. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Structural Similarities and Differences between Amyloidogenic and Non-Amyloidogenic Islet Amyloid Polypeptide (IAPP) Sequences and Implications for the Dual Physiological and Pathological Activities of These Peptides

IAPP, a 37 amino-acid peptide hormone belonging to the calcitonin family, is an intrinsically disordered protein that is coexpressed and cosecreted along with insulin by pancreatic islet β-cells in response to meals. IAPP plays a physiological role in glucose regulation; however, in certain species, IAPP can aggregate and this process is linked to β-cell death and Type II Diabetes. Using replica exchange molecular dynamics with extensive sampling (16 replicas per sequence and 600 ns per replica), we investigate the structure of the monomeric state of two species of aggregating peptides (human and cat IAPP) and two species of non-aggregating peptides (pig and rat IAPP). Our simulations reveal that the pig and rat conformations are very similar, and consist of helix-coil and helix-hairpin conformations. The aggregating sequences, on the other hand, populate the same helix-coil and helix-hairpin conformations as the non-aggregating sequence, but, in addition, populate a hairpin structure. Our exhaustive simulations, coupled with available peptide-activity data, leads us to a structure-activity relationship (SAR) in which we propose that the functional role of IAPP is carried out by the helix-coil conformation, a structure common to both aggregating and non-aggregating species. The pathological role of this peptide may have multiple origins, including the interaction of the helical elements with membranes. Nonetheless, our simulations suggest that the hairpin structure, only observed in the aggregating species, might be linked to the pathological role of this peptide, either as a direct precursor to amyloid fibrils, or as part of a cylindrin type of toxic oligomer. We further propose that the helix-hairpin fold is also a possible aggregation prone conformation that would lead normally non-aggregating variants of IAPP to form fibrils under conditions where an external perturbation is applied. The SAR relationship is used to suggest the rational design of therapeutics for treating diabetes.