Peptide-membrane Interactions Research Articles

Biophysical and functional assays for viral membrane fusion peptides

Membrane fusion is a protein catalyzed biophysical reaction that involves the simultaneous intermixing of two phospholipid bilayers and of the aqueous compartments bound by their respective bilayers. In the case of enveloped virus fusogens, short hydrophobic or amphipathic fusion peptides that are components of the larger fusion complex are essential for the membrane merger event. The process of cell–cell membrane fusion and syncytium formation induced by the nonenveloped fusogenic orthoreoviruses is driven by the Fusion-Associated Small Transmembrane (FAST) proteins, which are similarly dependent on the action of fusion peptides. In this article, we describe some simple methods for the biophysical characterization of viral membrane fusion peptides. Liposomes serve as an ideal model system for characterizing peptide–membrane interactions because their size, shape and composition can be readily manipulated. We present details of fluorescence assays used to elucidate the kinetics of membrane fusion as well as complimentary assays used to characterize peptide-induced liposome binding and aggregation.

Chemical Conjugation of the Neuropeptide Kyotorphin and Ibuprofen Enhances Brain Targeting and Analgesia

The pharmaceutical potential of natural analgesic peptides is mainly hampered by their inability to cross the blood-brain barrier, BBB. Increasing peptide-cell membrane affinity through drug design is a promising strategy to overcome this limitation. To address this challenge, we grafted ibuprofen (IBP), a nonsteroidal anti-inflammatory drug, to kyotorphin (l-Tyr-l-Arg, KTP), an analgesic neuropeptide unable to cross BBB. Two new KTP derivatives, IBP-KTP (IbKTP-OH) and IBP-KTP-amide (IbKTP-NH(2)), were synthesized and characterized for membrane interaction, analgesic activity and mechanism of action. Ibuprofen enhanced peptide-membrane interaction, endowing a specificity for anionic fluid bilayers. A direct correlation between anionic lipid affinity and analgesic effect was established, IbKTP-NH(2) being the most potent analgesic (from 25 μmol · kg(-1)). In vitro, IbKTP-NH(2) caused the biggest shift in the membrane surface charge of BBB endothelial cells, as quantified using zeta-potential dynamic light scattering. Our results suggest that IbKTP-NH(2) crosses the BBB and acts by activating both opioid dependent and independent pathways.

Dermaseptins as Models for the Elucidation of Membrane-Acting Helical Amphipathic Antimicrobial Peptides

Antimicrobial peptides (AMPs) produced by a wide variety of organisms are major actors of the host defense systems against invading pathogenic microorganisms. These peptides exhibit a broad spectrum of action against bacteria, yeasts, fungi, protozoa and viruses. It is widely believed that a large part of their antimicrobial effect derives from direct interactions with the lipid membrane surrounding the target cells, causing its permeabilization and cell lysis. However, the exact nature of these interactions is presently unclear. The skin of the amphibians has proved to be a remarkably rich storehouse of AMPs that encompass a wide variety of structural motifs. This natural AMP bank is used in combined approaches, based on biophysical and cellular biology methods, to elucidate how these peptides perturb the membrane and whether such membrane perturbations are related to the antimicrobial activity of these peptides. Here we review our current knowledge about the structure and the mechanism of action of the dermaseptin super-family, α-helical amphipathic AMPs isolated from the skin of frogs of the Phyllomedusa genus. Dermaseptins are genetically related, with a remarkable identity in signal sequences and acidic propieces of their preproforms but have clearly diverged to yield several families of microbicidal cationic peptides that are structurally distinct. Particularly, we focused on the orthologous peptides dermaseptin S and B of which the shortening from the carboxy terminal extremity causes a drastic change in their membrane disruption activity. These peptides could be good models to study the membrane-peptide interactions discussed in this review.

Investigating the effect of a single glycine to alanine substitution on interactions of antimicrobial peptide latarcin 2a with a lipid membrane

Latarcins are linear, α-helical antimicrobial peptides purified from the venom of the Central Asian spider Lachesana tarabaevi, with lytic activity against Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations. In this work, we investigated the role of the hinge in latarcin 2a (ltc2a, GLFGKLIKKFGRKAISYAVKKARGKH-COOH), which adopts a helix-hinge-helix conformation in membrane-mimicking environments, on peptide-membrane interactions and its potential effect on the selective toxicity of the peptide. A modified latarcin 2a, ltc2aG11A, obtained by replacing the glycine at position 11 with alanine (ltc2aG11A, GLFGKLIKKFARKAISYAVKKARGKH-COOH), adopts a more rigid structure due to the reduced conformational flexibility. Langmuir monolayer measurements combined with atomic force microscopy and X-ray photoemission electron microscopy (X-PEEM) indicate that both peptides bind and insert preferentially into anionic compared with zwitterionic phospholipid monolayers. Modified ltc2aG11A was found to be more disruptive of supported phospholipid bilayer modeling mammalian cell membrane. However, no considerable difference in lytic activity of the two peptides toward bacterial membrane was found. Overall the data indicate that decrease in the flexibility of ltc2a induced by the modification in the hinge region is likely to increase the peptide's nonspecific interactions with zwitterionic cell membranes and potentially increase its toxicity against eukaryotic cells.

Spatiotemporal Multicolor Labeling of Individual Cells Using Peptide-Functionalized Quantum Dots and Mixed Delivery Techniques

Multicolor fluorescent labeling of both intra- and extracellular structures is a powerful technique for simultaneous monitoring of multiple complex biochemical processes. This approach remains extremely challenging, however, as it often necessitates the combinatorial use of numerous targeting probes (e.g., antibodies), multistep bioconjugation chemistries, different delivery strategies (e.g., electroporation or transfection reagents), cellular fixation coupled with membrane permeabilization, and complex spectral deconvolution. Here, we present a nanoparticle-based fluorescence labeling strategy for the multicolor labeling of distinct subcellular compartments within live cells without the need for antibody conjugation or cellular fixation/permeabilization. This multipronged approach incorporates an array of delivery strategies, which localize semiconductor quantum dots (QDs) to various subcellular structures. QD uptake is implemented in a spaciotemporal manner by staggering the delivery of QD-peptide composites and exploiting various innate (peptide-mediated endocytosis, peptide-membrane interaction, polymer-based transfection) along with physical (microinjection) cellular delivery modalities to live cells growing in culture over a 4 day period. Imaging of the different intracellular labels is simplified by the unique photophysical characteristics of the QDs in combination with Förster resonance energy transfer sensitization, which allow for multiple spectral windows to be accessed with one excitation wavelength. Using this overall approach, QDs were targeted to both early and late endosomes, the cellular cytosol, and the plasma membrane in live cells, ultimately allowing for simultaneous five-color fluorescent imaging.

Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides

To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

Multiple Peptide Resistance Factor (MprF)-mediated Resistance of Staphylococcus aureus against Antimicrobial Peptides Coincides with a Modulated Peptide Interaction with Artificial Membranes Comprising Lysyl-Phosphatidylglycerol

Modification of the membrane lipid phosphatidylglycerol (PG) of Staphylococcus aureus by enzymatic transfer of a l-lysine residue leading to lysyl-PG converts the net charge of PG from -1 to +1 and is thought to confer resistance to cationic antimicrobial peptides (AMPs). Lysyl-PG synthesis and translocation to the outer leaflet of the bacterial membrane are achieved by the membrane protein MprF. Consequently, mutants lacking a functional mprF gene are in particular vulnerable to the action of AMPs. Hence, we aim at elucidating whether and to which extent lysyl-PG modulates membrane binding, insertion, and permeabilization by various AMPs. Lysyl-PG was incorporated into artificial lipid bilayers, mimicking the cytoplasmic membrane of S. aureus. Moreover, we determined the activity of the peptides against a clinical isolate of S. aureus strain SA113 and two mutants lacking a functional mprF gene and visualized peptide-induced ultrastructural changes of bacteria by transmission electron microscopy. The studied peptides were: (i) NK-2, an α-helical fragment of mammalian NK-lysin, (ii) arenicin-1, a lugworm β-sheet peptide, and (iii) bee venom melittin. Biophysical data obtained by FRET spectroscopy, Fourier transform infrared spectroscopy, and electrical measurements with planar lipid bilayers were correlated with the biological activities of the peptides. They strongly support the hypothesis that peptide-membrane interactions are a prerequisite for eradication of S. aureus. However, degree and mode of modulation of membrane properties such as fluidity, capacitance, and conductivity were unique for each of the peptides. Altogether, our data support and underline the significance of lysyl-PG for S. aureus resistance to AMPs.

Transmembrane Pores Formed by Human Antimicrobial Peptide LL-37

Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the α-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 23–33 Å. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.

Exploring Peptide-Membrane Interactions with Coarse-Grained MD Simulations

The interaction of α-helical peptides with lipid bilayers is central to our understanding of the physicochemical principles of biological membrane organization and stability. Mutations that alter the position or orientation of an α-helix within a membrane, or that change the probability that the α-helix will insert into the membrane, can alter a range of membrane protein functions. We describe a comparative coarse-grained molecular dynamics simulation methodology, based on self-assembly of a lipid bilayer in the presence of an α-helical peptide, which allows us to model membrane transmembrane helix insertion. We validate this methodology against available experimental data for synthetic model peptides (WALP23 and LS3). Simulation-based estimates of apparent free energies of insertion into a bilayer of cystic fibrosis transmembrane regulator-derived helices correlate well with published data for translocon-mediated insertion. Comparison of values of the apparent free energy of insertion from self-assembly simulations with those from coarse-grained molecular dynamics potentials of mean force for model peptides, and with translocon-mediated insertion of cystic fibrosis transmembrane regulator-derived peptides suggests a nonequilibrium model of helix insertion into bilayers.

Hydrophobicity drives the cellular uptake of short cationic peptide ligands

Short cationic linear peptide analogs (LPAs, prepared as Arg-C( n )-Arg-C( n )-Lys, where C( n ) represents an alkyl linkage with n = 4, 7 or 11) were synthesized and tested in human breast carcinoma BT-20 and CCRF-CEM leukemia cells for their application as targeting ligands. With constant LPA charge (+4), increasing the alkyl linkage increases the hydrophobic/hydrophilic balance and provides a systematic means of examining combined electrostatic and hydrophobic peptide-membrane interactions. Fluorescently conjugated LPA-C(11) (F-LPA-C(11)) demonstrated significant uptake, whereas there was negligible uptake of the shorter LPAs. By varying temperature (4°C and 37°C) and cell type, the results suggest that LPA-C(11) internalization is nonendocytic and nonspecific. The effect of LPA binding on the phase behavior, structure, and permeability of model membranes composed of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine (DPPC/DPPS, 85/15) was studied using differential scanning calorimetry (DSC), cryogenic transmission electron microscopy (cryo-TEM), and fluorescence leakage studies to gain insight into the LPA uptake mechanism. While all LPAs led to phase separation, LPA-C(11), possessing the longest alkyl linkage, was able to penetrate into the bilayer and caused holes to form, which led to membrane disintegration. This was confirmed by rapid and complete dye release by LPA-C(11). We propose that LPA-C(11) achieves uptake by anchoring to the membrane via hydrophobicity and forming transient membrane voids. LPAs may be advantageous as drug transporter ligands because they are small, water soluble, and easy to prepare.

IR Spectroscopy of Protein and Peptide-Membrane Interactions: In Situ Strategies for Tracking Fusion, Remodelling, and Refolding

Attenuated total reflection-infrared spectroscopy is a powerful technique for characterizing protein and peptide-membrane interactions. Using a real-time, flow-through design and a single bounce diamond ATR element, we have examined how three different families of proteins interact with model supported lipid bilayers. These include (1) a lipid-remodelling protein from the saposin family; (2) the dynamin-related GTP-ase Mgm1p; (3) a series of model membranolytic peptides designed to adopt a helical motif in bacterial membranes. Our studies provided compelling evidence of the action of these proteins, including membrane remodeling, GTP-ase activity, and peptide aggregation. We will describe the integration of laser-based fluorescence spectroscopy with ATR-IR, which, when coupled with correlated in situ atomic force microscopy, will afford a uniquely capable multimodal imaging and characterization platform.

In Situ Mapping of Membranolytic Peptide-Membrane Interactions by Coupled Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy-Atomic Force Microscopy (ATR-FTIR-AFM)

The mechanisms by which membranolytic peptides may exert their action are highly complex and specific. They may involve peptide self-association, membrane-induced refolding, or targeting of specific lipid domains or components. Insights into these behaviours are critical for the de novo design of peptide-based antimicrobial agents. To better understand these mechanisms, we have applied coupled attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR)-atomic force microscopy (AFM) to directly visualize and characterize the interactions of two different classes of model membranolytic peptides with supported planar lipid bilayers. Our studies provided direct evidence of membrane rearrangement and peptide aggregation by a de novo cationic antimicrobial peptide designed to adopt a helical motif in bacterial membranes. We also obtained in situ evidence of specific secondary structure motifs associated with a membrane-induced fibrillization of peptides derived from haemolytic proteins obtained from sea anemone. This coupled approach provides a unique opportunity to directly link spectroscopic details associated with peptide-membrane interactions with in situ structural insights obtained on nanometer length scales.

Conformational Characterization of Tachykinin Neuropeptides: Role of the Polyproline II Structure

Substance P (SP), Neurokinin A (NKA) and Scyliorhinin I (Scyl) peptides belong to the Tachykinin family and are agonists for Neurokinin1 (NK1) GPCR receptor with different potent activities. These Tachykinins are involved in several physiological processes and some neurodegenerative disorders, what make them relevant therapeutically agents. The characterization of the active peptide conformations of the Tachykinins is essential for the elucidation of the mechanism of action and for the design of drugs. To address the molecular basis for the peptide recognition by the NK1 receptor we studied the conformation of SP, NKA and Scyl in solution and membrane-mimic environments: micelles and liposomes, by using CD and FTIR spectroscopies. The analysis of CD data revealed that the three peptides form a partially α helical structure in the presence of the negatively charged micelles and vesicles, but not in zwitterionic DMPC. By performing CD spectra at increasing temperatures we demonstrated that in an aqueous environment SP, NKA and Scyl form extended polyproline II (PPII) helical structure. The same structure was found in the membrane mimics, which do not induce formation of α helical conformation of the peptides. FTIR experiments performed in D2O support the presence of PPII conformation. Further we questioned whether the formation of PPII needs peptide binding to the membrane or it is a peptide intrinsic property, by using fluorescence techniques. We propose that the peptide-membrane interaction is a two-step process involving first electrostatic interactions through PPII structure, followed by the folding and insertion of the alpha-helical segment.

Membrane Interactions of Antimicrobial Retro-Lactoferricin Peptides by Solid-State NMR and Partitioning Assays

Bovine lactoferricin (LfB25) is a 25-residue peptide released from the N-terminal of bovine lactoferrin which exhibits broad spectrum antimicrobial properties. A hexapeptide, LfB6 (RRWQWR-NH2), is a small fragment that retains significant antimicrobial activity (Tomita et al. 1994. Acta Paediatr Jpn. 36:585). We seek to modify and understand the antimicrobial properties of LfB6 through various modifications. Previous studies from our lab have shown that antimicrobial activity can be increased by N-acylation and Trp-methylation (Greathouse et al. 2008. J. Pept. Sci. 14:1103), as well as by reversal of the peptide sequence (Retro-LfB6). Acylation and Trp-methylation enhance the binding of the peptides to bacterial-like membranes. In addition, Trp-methylation allows for selective ring labeling with deuterium for solid-state NMR studies. To determine whether combining the three modifications (sequence reversal, Trp methylation, and acylation) will further enhance activity, two Retro-LfB6 peptides have been synthesized with a methylated Trp at sequence position 4. One of the peptides has been further modified by N-acylation with a 6-carbon fatty acid. Retro-LfB6 (RWQMeWRR-NH2) and C6 Retro-LfB6 (C6-RWQMeWRR-NH2) were examined by solid-state 31P and 2H NMR in mechanically aligned bilayers composed of a neutral membrane with a zwitterionic head group (DMPC) and a bacterial-like membrane composed of a 3:1 mixture of neutral (DMPC) and anionic lipids (DMPG). 2H NMR spectra suggest weaker binding or less well-defined orientations of the Retro-LfB6 peptides compared to native LfB6 peptides. 31P NMR spectra reveal that the lipids remain primarily in a bilayer arrangement, with the peptides causing little change to the phosphate head groups. Antimicrobial assays demonstrate that the C6-acylated, Trp-methylated, Retro-LfB6 peptide has enhanced activity relative to the native peptide against S. aureus. Results from partitioning assays will be compared with the NMR and antimicrobial data.

Biophysical Characterization of Peptide Membrane Interactions and Membrane Permeabilization

Certain membrane-active amphipathic peptides, such as antimicrobial peptides, possess a characteristic feature of having several cationic amino acid residues at either N- or C-terminus. In an attempt to determine the functional significance of C-terminal cationic residues, we have studied the membrane binding mode and membrane pore formation by a 20-amino acid synthetic amphipathic peptide. Two cationic residues were replaced to Leu or Glu to see how the membrane binding and permeabilization activities of the peptide are modulated by charge neutralization and charge reversal mutations. The depth of membrane insertion of the wild type and mutant peptides was determined by measuring the quenching of the fluorescence of a single, C-terminally located tryptophan by dibromo-phosphatidylcholines brominated at various positions of the acyl chains, using pure POPC and 70% POPC + 30% POPG in lipid vesicles. In case of pure POPC membranes, Trp in all three peptides was located at around 9 Å from the membrane center, while for 30% POPG, Trp was inserted much deeper. Studies on peptide-mediated calcein release from phospholipid vesicles were conducted to see if the peptides are able to cause membrane pore formation and content release. Peptides with positively charged residues showed maximum calcein release for both lipid compositions whereas negative or hydrophobic residues were less effective in vesicle permeabilization. Preliminary ATR-FTIR experiments using both POPC and POPG have been performed to identify the orientation of the peptides within the lipid bilayer and their effect of the lipid order.

A Langmuir Approach Using Monolayer Interactions to Investigate Surface Active Peptides

The Langmuir Blodgett apparatus provides a versatile system for studying the interfacial properties of peptides and peptide-membrane interactions under controlled conditions. Using amphiphilic α-helical peptides to highlight studies undertaken, here we discuss the use of this system to provide information on the surface activity of peptides and describe the insights these studies give into biological function.

Monitoring membrane binding and insertion of peptides by two-color fluorescent label

Herein, we developed an approach for monitoring membrane binding and insertion of peptides using a fluorescent environment-sensitive label of the 3-hydroxyflavone family. For this purpose, we labeled the N-terminus of three synthetic peptides, melittin, magainin 2 and poly-l-lysine capable to interact with lipid membranes. Binding of these peptides to lipid vesicles induced a strong fluorescence increase, which enabled to quantify the peptide–membrane interaction. Moreover, the dual emission of the label in these peptides correlated well with the depth of its insertion measured by the parallax quenching method. Thus, in melittin and magainin 2, which show deep insertion of their N-terminus, the label presented a dual emission corresponding to a low polar environment, while the environment of the poly-l-lysine N-terminus was rather polar, consistent with its location close to the bilayer surface. Using spectral deconvolution to distinguish the non-hydrated label species from the hydrated ones and two photon fluorescence microscopy to determine the probe orientation in giant vesicles, we found that the non-hydrated species were vertically oriented in the bilayer and constituted the best indicators for evaluating the depth of the peptide N-terminus in membranes. Thus, this label constitutes an interesting new tool for monitoring membrane binding and insertion of peptides.

Analysis of membrane interactions of antibiotic peptides using ITC and biosensor measurements

The interaction of the lantibiotic gallidermin and the glycopeptide antibiotic vancomycin with bacterial membranes was simulated using mass sensitive biosensors and isothermal titration calorimetry (ITC). Both peptides interfere with cell wall biosynthesis by targeting the cell wall precursor lipid II, but differ clearly in their antibiotic activity against individual bacterial strains. We determined the binding affinities of vancomycin and gallidermin to model membranes ± lipid II in detail. Both peptides bind to DOPC/lipid II membranes with high affinity (K D 0.30 μM and 0.27 μM). Gallidermin displayed also strong affinity to pure DOPC membranes (0.53 μM) an effect that was supported by ITC measurements. A surface acoustic wave (SAW) sensor allowed measurements in the picomolar concentration range and revealed that gallidermin targets lipid II at an equimolar ratio and simultaneously inserts into the bilayer. These results indicate that gallidermin, in contrast to vancomycin, combines cell wall inhibition and interference with the bacterial membrane integrity for potent antimicrobial activity.

Fluctuations and the Rate-Limiting Step of Peptide-Induced Membrane Leakage

Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.

The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12-18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.