Conformational and self-assembly studies of helix forming hexapeptides containing two alpha-amino isobutyric acids

Single crystal X-ray diffraction studies reveal that three hexapeptides with general formula Boc-Ile-Aib-Xx-Ile-Aib-Yy-OMe, where Xx and Yy are Leu in peptide I, Len and Phe in peptide II, and Phe and Leu in peptide III, respectively, adopt equivalent conformations that can be described as mixed 3(10)/alpha-helice with two 4 -> 1 and two 5 -> 1 intramolecular N-H center dot center dot center dot O=C H-bonds. The peptides do not generate any helixterminating Schellman motif despite having Aib at the penultimate position from C-terminus. In the crystalline state, the helices are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. The CD Studies of the three hexapeptides in acetonitrile indicate that they are folded in well-developed 3(10)-helical structures. NMR studies of peptide I in CDCl3 also suggest the formation of a homogeneous 3 m-helical structure. The field emission scanning electron microscopic (FE-SEM) images of peptide 11 in the solid state reveal a non-twisted ribbon-like morphology, which is formed through lateral association of non-twisted filaments. (c) 2007 Elsevier Ltd. All rights reserved.

Stabilization of two smallest possible diastereomeric beta-hairpins in a water soluble tetrapeptide containing non-coded alpha-amino isobutyric acid (Aib) and m-amino benzoic acid

Single crystal X-ray diffraction study reveals that the water soluble tetrapeptide H2N-Ile-Aib-Leu-m-ABA-CO2H, containing non-coded Aib (alpha-amino isobutyric acid) and m-ABA (meta-amino benzoic acid), crystallizes with two smallest possible diastereomeric beta-hairpin molecules in the asymmetric unit. Although in both of the molecules the chiralities at Ile(1) and Leu(3) are S, a conformational reversal in the back bone chain is observed to produce the beta-hairpins with beta-turn conformations of type II and II'. Interestingly Aib which is known to adopt helical conformation, adopts unusual semi-extended conformation with phi: -49.5(5)degrees, psi: 135.2(5)degrees in type II and phi: 50.6(6)degrees. psi: -137.0(4)degrees in type II' for occupying the i + 1 position of the beta-turns. The two hairpin molecules are further interlocked through intermolecular hydrogen bonds and electrostatic interactions between CO2- and -+NH3 groups to form dimeric supramolecular beta-hairpin aggregate in the crystal state. The CD measurement and 2D NMR study of the peptide in aqueous medium support the existence of beta-hairpin structure in water. (C) 2009 Elsevier B.V. All rights reserved.

A new molecular scaffold for the formation of supramolecular peptide double helices: the crystallographic insight

A series of water-soluble synthetic dipeptides (1-3) with an N-terminally located beta-alanine residue, beta-alanyl-L-valine (1), beta-alanyl-L-isoleucine (2), and beta-alanyl-L-phenylalanine (3, form hydrogen-bonded supramolecular double helices with a pitch length of 1 nm, whereas the C-terminally positioned beta-alanine containing dipeptide (4), L-phenylalanyl-beta-alanine, does not form a supramolecular double helical structure. beta-Ala-Xaa (Xaa = Val/Ile/Phe) can be regarded as a new motif for the formation of supramolecular double helical structures in the solid state.

Overlapping double turn conformations adopted by tetrapeptides containing non-coded alpha-Amino Isobutyric Acid (AIB) and formation of tape-like structures through supramolecular helix mediated self-assembly

Single crystal X-ray diffraction studies and solvent dependent H-1 NMR titrations reveal that a set of four tetrapeptides with general formula Boc-Xx(1)-Aib(2)-Yy(3)-Zz(4)-OMe, where Xx, Yy and Zz are coded L- amino acids, adopt equivalent conformations that can be described as overlapping double turn conformations stabilized by two 4 -> 1 intramolecular hydrogen bonds between Yy(3)-NH and Boc C=O and Zz(4)-NH and Xx(1)C=O. In the crystalline state, the double turn structures are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. Field emission scanning electron microscopic (FE-SEM) images of the tetrapeptides in the solid state reveal that they can form flat tape-like structures. The results establish that synthetic Aib containing supramolecular helices can form highly ordered self-aggregated amyloid plaque like human amylin.

Supramolecular beta-sheet and nanofibril formation by self-assembling tripeptides containing an N-terminally located gamma-aminobutyric acid residue

Three terminally protected tripeptides Boc-gamma-Abu-Val-Leu-OMe 1, Boc-gamma-Abu-Leu-Phe-OMe 2 and Boc-gamma-Abu-Val-Tyr-OMe 3 (gamma-Abu = gamma-aminobutyric acid) each containing an N-terminally positioned gamma-aminobutyric acid residue have been synthesized, purified and studied. FT-IR studies of all these peptides revealed that these peptides form intermolecularly hydrogen bonded supramolecular beta-sheet structures. Peptides 1, 2 and 3 adopt extended backbone beta-strand molecular structures in crystals. Crystal packing of all these peptides demonstrates that these beta-strand structures self-assemble to form intermolecularly H-bonded parallel beta-sheet structures. Peptide 3 uses a side chain tyrosyl -OH group as an additional hydrogen bonding functionality in addition to the backbone CONH groups to pack in crystals. Transmission electron microscopic studies of all peptides indicate that they self-assemble to form nanofibrillar structures of an average diameter of 65 nm. These peptide fibrils exhibit amyloid-like behavior as they bind to a physiological dye Congo red and show a characteristic green-gold birefringence under polarizing microscope.

Self-assembly of an amyloid peptide fragment–PEG conjugate: lyotropic phase formation and influence of PEG crystallization

The self-assembly of PEGylated peptides containing a modified sequence from the amyloid beta peptide, YYKLVFF, has been studied in aqueous solution. Two PEG molar masses, PEG1k and PEG3k, were used in the conjugates. It is shown that both YYKLVFF–PEG hybrids form fibrils comprising a peptide core and a PEG corona. The fibrils are much longer for YYKLVFF–PEG1k, pointing to an influence of PEG chain length. The beta-sheet secondary structure of the peptide is retained in the conjugate. Lyotropic liquid crystal phases, specifically nematic and hexagonal columnar phases, are formed at sufficiently high concentration. Flow alignment of these mesophases was investigated by small-angle neutron scattering with in situ steady shearing in a Couette cell. On drying, PEG crystallization occurs leading to characteristic peaks in the X-ray diffraction pattern, and to lamellar structures imaged by atomic force microscopy. The X-ray diffraction pattern retains features of the cross-beta pattern from the beta-sheet structure, showing that this is not disrupted by PEG crystallization.

Amyloid-like fibrillogenesis through supramolecular helix-mediated self-assembly of tetrapeptides containing non-coded alpha-aminoisobutyric acid (Aib) and 3-aminobenzoic acid (m-ABA)

Single-crystal X-ray diffraction studies of two terminally protected tetrapeptides Boc-Ile-Aib-Val-m-ABA-OMe (I) and Boc-Ile-Aib-Phe-m-ABA-OMe (II) (Aib = alpha-aminoisobutyric acid; m-ABA = meta-aminobenzoic acid) reveal that they form continuous H-bonded helices through the association of double-bend (type III and I) building blocks. NMR Studies support the existence of the double-bend (type Ill and I) structures of the peptides in solution also. Field emission scanning electron-microscopic (FE-SEM) and high-resolution transmission electron-microscopic (HR-TEM) images of the peptides exhibit amyloid-like fibrils in the solid state. The Congo red-stained fibrils of peptide I and II, observed between crossed polarizers, show green-gold birefringence, a characteristic of amyloid fibrils.

Influence of end-capping on the self-assembly of model amyloid peptide fragments

The influence of charge and aromatic stacking interactions on the self-assembly of a series of four model amyloid peptides has been examined. The four model peptides are based on the KLVFF motif from the amyloid Beta peptide, ABeta(16-20) extended at the N terminus with two Beta-alanine residues. We have studied NH2-BetaABetaAKLVFF-COOH (FF), NH2-BetaABetaAKLVFCOOH (F), CH3CONH-BetaABetaAKLVFF-CONH2 (CapF), and CH3CONH-BetaABetaAKLVFFCONH2 (CapFF). The former two are uncapped (net charge plus 2) and differ by one hydrophobic phenylalanine residue; the latter two are the analogous capped peptides (net charge plus 1). The self-assembly characteristics of these peptides are remarkably different and strongly dependent on concentration. NMR shows a shift from carboxylate to carboxylic acid forms upon increasing concentration. Saturation transfer measurements of solvent molecules indicate selective involvement of phenylalanine residues in driving the self-assembly process of CapFF due presumably to the effect of aromatic stacking interactions. FTIR spectroscopy reveals beta-sheet features for the two peptides containing two phenylalanine residues but not the single phenylalanine residue, pointing again to the driving force for self-assembly. Circular dichroism (CD) in dilute solution reveals the polyproline II conformation, except for F which is disordered. We discuss the relationship of this observation to the significant pH shift observed for this peptide when compared the calculated value. Atomic force microscopy and cryogenic-TEM reveals the formation of twisted fibrils for CapFF, as previously also observed for FF. The influence of salt on the self-assembly of the model beta-sheet forming capped peptide CapFF was investigated by FTIR. Cryo-TEM reveals that the extent of twisting decreases with increased salt concentration, leading to the formation of flat ribbon structures. These results highlight the important role of aggregation-induced pKa shifts in the self-assembly of model beta-sheet peptides.

Modeling the tetraphenylalanine-PEG hybrid amphiphile: from DFT calculations on the peptide to molecular dynamics simulations on the conjugate

The conformational properties of the hybrid amphiphile formed by the conjugation of a hydrophobic peptide with four phenylalanine (Phe) residues and hydrophilic poly(ethylene glycol), have been investigated using quantum mechanical calculations and atomistic molecular dynamics simulations. The intrinsic conformational preferences of the peptide were examined using the building-up search procedure combined with B3LYP/ 6-31G(d) geometry optimizations, which led to the identification of 78, 78, and 92 minimum energy structures for the peptides containing one, two, and four Phe residues. These peptides tend to adopt regular organizations involving turn-like motifs that define ribbon or helicallike arrangements. Furthermore, calculations indicate that backbone ... side chain interactions involving the N-H of the amide groups and the pi clouds of the aromatic rings play a crucial role in Phe-containing peptides. On the other hand,MD simulations on the complete amphiphile in aqueous solution showed that the polymer fragment rapidly unfolds maximizing the contacts with the polar solvent, even though the hydrophobic peptide reduce the number of waters of hydration with respect to an individual polymer chain of equivalent molecular weight. In spite of the small effect of the peptide in the hydrodynamic properties of the polymer, we conclude that the two counterparts of the amphiphile tend to organize as independent modules.

Modulating self-assembly of a nanotape-forming peptide amphiphile with an oppositely charged surfactant

A peptide amphiphile (PA) C16-KTTKS, containing a pentapeptide headgroup based on a sequence from procollagen I attached to a hexadecyl lipid chain, self-assembles into extended nanotapes in aqueous solution. The tapes are based on bilayer structures, with a 5.2 nm spacing. Here, we investigate the effect of addition of the oppositely charged anionic surfactant sodium dodecyl sulfate (SDS) via AFM, electron microscopic methods, small-angle X-ray scattering and X-ray diffraction among other methods. We show that addition of SDS leads to a transition from tapes to fibrils, via intermediate states that include twisted ribbons. Addition of SDS is also shown to enhance the development of remarkable lateral ‘‘stripes’’ on the nanostructures, which have a 4 nm periodicity. This is ascribed to counterion condensation. The transition in the nanostructure leads to changes in macroscopic properties, in particular a transition from sol to gel is noted on increasing SDS (with a further reentrant transition to sol on further increase of SDS concentration). Formation of a gel may be useful in applications of this PA in skincare applications and we show that this can be controlled via development of a network of fine stranded fibrils.

Influence of a non-ionic amphiphilic copolymer on the self-assembly of a peptide amphiphile that forms nanotapes

The influence of a non-ionic polymeric surfactant on the self-assembly of a peptide amphiphile (PA) that forms nanotapes is investigated using a combination of microscopic, scattering and spectroscopic techniques. Mixtures of Pluronic copolymer P123 with the PA C16-KTTKS in aqueous solution were studied at a fixed concentration of the PA at which it is known to self-assemble into extended nanotapes, but varying P123 concentration. We find that P123 can disrupt the formation of C16- KTTKS nanotapes, leading instead to cylindrical nanofibril structures. The spherical micelles formed by P123 at room temperature are disrupted in the presence of the PA. There is a loss of cloudiness in the solutions as the large nanotape aggregates formed by C16-KTTKS are broken up, by P123 solubilization. At least locally, b-sheet structure is retained, as confirmed by XRD and FTIR spectroscopy, even for solutions containing 20 wt% P123. This indicates, unexpectedly, that peptide secondary structure can be retained in solutions with high concentration of non-ionic surfactant. Selfassembly in this system exhibits slow kinetics towards equilibrium, the initial self-assembly being dependent on the order of mixing. Heating above the lipid chain melting temperature assists in disrupting trapped non-equilibrium states.

Slow-release RGD-peptide hydrogel monoliths

We report on the formation of hydrogel monoliths formed by functionalized peptide Fmoc-RGD (Fmoc: fluorenylmethoxycarbonyl) containing the RGD cell adhesion tripeptide motif. The monolith is stable in water for nearly 40 days. The gel monoliths present a rigid porous structure consisting of a network of peptide fibers. The RGD-decorated peptide fibers have a β-sheet secondary structure. We prove that Fmoc-RGD monoliths can be used to release and encapsulate material, including model hydrophilic dyes and drug compounds. We provide the first insight into the correlation between the absorption and release kinetics of this new material and show that both processes take place over similar time scales.

Interaction between a cationic surfactant-like peptide and lipid vesicles and its relationship to antimicrobial activity

We investigate the properties of an antimicrobial surfactant-like peptide (Ala)6(Arg), A6R, containing a cationic headgroup. The interaction of this peptide with zwitterionic (DPPC) lipid vesicles is investigated using a range of microscopic, X-ray scattering, spectroscopic, and calorimetric methods. The β-sheet structure adopted by A6R is disrupted in the presence of DPPC. A strong effect on the small-angle X-ray scattering profile is observed: the Bragg peaks from the DPPC bilayers in the vesicle walls are eliminated in the presence of A6R and only bilayer form factor peaks are observed. All of these observations point to the interaction of A6R with DPPC bilayers. These studies provide insight into interactions between a model cationic peptide and vesicles, relevant to understanding the action of antimicrobial peptides on lipid membranes. Notably, peptide A6R exhibits antimicrobial activity without membrane lysis.

Tuning self-assembled nanostructures through enzymatic degradation of a peptide amphiphile

The enzymatic cleavage of a peptide amphiphile (PA) is investigated. The self-assembly of the cleaved products is distinct from that of the PA substrate. The PA C16-KKFFVLK is cleaved by α-chymotrypsin at two sites leading to products C16-KKF with FVLK and C16-KKFF with VLK. The PA C16-KKFFVLK forms nanotubes and helical ribbons at room temperature. Both PAs C16-KKF and C16-KKFF corresponding to cleavage products instead self-assemble into 5-6 nm diameter spherical micelles, while peptides FVLK and VLK do not adopt well-defined aggregate structures. The secondary structures of the PAs and peptides are examined by FTIR and circular dichroism spectroscopy and X-ray diffraction. Only C16-KKFFVLK shows substantial β-sheet secondary structure, consistent with its self-assembly into extended aggregates, based on PA layers containing hydrogen-bonded peptide headgroups. This PA also exhibits a thermoreversible transition to twisted tapes on heating.

Self-assembly of a Model Peptide incorporating a Hexa-Histidine sequence attached to an Oligo-Alanine sequence, and binding to Gold NTA/Nickel nanoparticles

Amyloid fibrils are formed by a model surfactant-like peptide (Ala)10-(His)6 containing a hexahistidine tag. This peptide undergoes a remarkable two-step self-assembly process with two distinct critical aggregation concentrations (cac’s), probed by fluorescence techniques. A micromolar range cac is ascribed to the formation of prefibrillar structures, whereas a millimolar range cac is associated with the formation of well-defined but more compact fibrils. We examine the labeling of these model tagged amyloid fibrils using Ni-NTA functionalized gold nanoparticles (Nanogold). Successful labeling is demonstrated via electron microscopy imaging. The specificity of tagging does not disrupt the β-sheet structure of the peptide fibrils. Binding of fibrils and Nanogold is found to influence the circular dichroism associated with the gold nanoparticle plasmon absorption band. These results highlight a new approach to the fabrication of functionalized amyloid fibrils and the creation of peptide/nanoparticle hybrid materials.