Effects of the sequence and size of non-polar residues on self-assembly of amphiphilic peptides

Peptides with alternating hydrophobic and polar amino acids have been shown to form stable beta-sheet secondary structures and self-assemble into hydrogel-like matrices in the presence of physiological salt concentrations. We hypothesized that the sequence and steric size differences of non-polar residues can affect the balance of peptide intermolecular forces in solution that drive self-assembly. To test this hypothesis, we designed a library of artificial amphiphilic peptides based on the sequence (FEFEFKFK)2 by substituting combinations of the non-polar residues glycine, alanine, valine, leucine and isoleucine for phenylalanine. Peptide structure and self-assembly were characterized using scanning electron microscopy, the Thioflavin T assay, transmission electron microscopy, X-ray fiber diffraction and circular dichroism spectroscopy. The sequence and steric size of non-polar residues are shown to cause variations in peptide secondary structures and create significant differences in the matrix morphology of self-assembled peptides.     

Interaction of substance P with tubulin

Binding of the peptide neurotransmitter substance P to brain tubulin in vitro inhibits self-assembly of the protein into microtubules and disrupts preassembled microtubules. This cooperative inhibition of the maximum extent of self-assembly by substance P is explicable in terms of preferential binding to the protomer state as compared to the polymer state of tubulin. The inhibition is relieved by the microtubule-associated protein MAP2, which evidently acts in a mixed competitive-noncompetitive fashion. Substance P interacts directly with the isolated C-terminal 4-kDa peptide fragment of tubulin, which appears to contain the specific binding area for MAP2, but is without effect on the self-assembly of the larger (48-kDa) part of the tubulin molecule called S-tubulin. The results are consistent with the C-terminal fragment having a binding site for the cationic substance P as well as for MAP2. However, factors other than electrostatic interaction must be operative, since the sulfoxide of substance P, a derivative with oxidized methionine but similar electrostatic characteristics, is inactive in inhibiting the extent of microtubule assembly.     

Characterization of a membrane-specific tubulin isoform by peptide mapping

A membrane-specific tubulin-like protein, found in preparations of synaptic plasma membranes and brain mitochondria, was analyzed by chemical and proteolytic peptide mapping to determine which part of the molecule was different from cytoplasmic tubulin. The membrane polypeptide was identical to alpha tubulin in the first two-thirds of the molecule containing the amino terminal, as found by peptide mapping. However, some differences were observed in the peptide maps of the carboxy terminal one third of the molecule which includes a domain that is important in the regulation of tubulin self-assembly.     

Circular dichroism and Fourier transform infrared spectroscopic studies on self-assembly of tetrapeptide derivative in solution and solvated film.

Aggregation of the hydrophobic peptide derivative Boc-Ala-Ile-Ile-Gly-OMe (1) was examined in methanol solution and in solvated film states. Formation of the peptide by self-assembly was evidenced using fluorescence [Mg salt of 8-anilino-naphthalenesulfonic acid (ANS) as an external probe] and circular dichroism (CD) spectroscopic techniques. In solution, peptide 1 formed as a stable aggregate at a concentration around 3 x 10(-4)m. The peptide gelled into a thin film for which we carried out CD and Fourier transform infrared (FTIR) measurements. Our spectroscopic study on peptide films at differing methanol concentrations indicates that the helical content of the peptide decreases with decreasing methanol concentration in solvated films. However, by reducing the methanol concentration we were able to observe a conformational transition from a predominantly helical turn to a beta-sheet structure via a random coil conformation. Our study focused on the aggregation of the alpha-helical turn-forming peptide derivative, which shows conformational transition on changing solvent concentration in the film form.     

Computer simulations of extractant primary amine N1923 and N1923 hydrochloride salt at water/chloroform interface

A molecular dynamics (MD) simulation has been carried out to investigate the structural and dynamical properties of extractant methyloctadecyl amine (N1923), and its hydrochloride salt at water/chloroform interface. The simulation results show that both N1923 molecule and protonated N1923 ion can self-assembly form a monolayer at the interface like most non-ionic or cationic surfactants. It is not only observed that the headgroup N atom of protonated N1923 ion arranges more tightly and orderly than that of N1923 molecule but also verified that the protonated headgroup of N1923 ion has stronger hydration ability than the polar headgroup of N1923 molecule.     

超分子多肽自组装在生物医学中的应用

近年来,自组装多肽纳米技术因其可形成规则有序的结构、具有多样的功能而备受关注。研究发现自组装多肽能在特定的条件下形成具有确定结构的聚集体,这种聚集体具备生物相容性好、稳定性高等优点,表现出不同于单体多肽分子的特性和优势,因此其在药物传递、组织工程、抗菌等领域具有良好的应用前景。文中介绍了自组装多肽形成的分子机理、类型、影响因素,综述了自组装多肽形成的纤维肽基水凝胶与自组装抗菌肽的最新进展,并提出目前多肽自组装技术所存在的问题及展望。     

Morphology of artificial silica matrices formed via autosilification of a silaffin/protein polymer chimera

Protein polymers (long-chain proteins in which a specific amino acid sequence "monomer" is repeated through the molecule) are found widely in nature, and these materials exhibit a diverse array of physical properties. One class of self-assembling proteins is hydrophobic-polar (HP) protein polymers capable of self-assembly under the appropriate solution conditions. We generated a chimeric protein consisting of an HP protein polymer monomer unit, EAK 1 (sequence n-AEAEAKAKAEAEAKAK-c), and a silaffin peptide, R5 (sequence: n-SSKKSGSYSGSKGSKRRIL-c). First identified in diatoms, silaffins represent a class of proteins and peptides capable of directing silica precipitation in vitro at neutral pH and ambient temperatures. The EAK 1-R5 chimera demonstrated self-assembly into hydrogels and the ability to direct silica precipitation in vitro. This chimera is capable of generating silica morphologies and feature sizes significantly different from those achievable with the R5 peptide alone, indicating that fusions of silaffins with self-assembling proteins may be a route to controlling the morphology of artificially produced silica matrices.     

Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering

Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.     

Molecular mechanisms of peptide loading by the tumor rejection antigen/heat shock chaperone gp96 (GRP94)

Complexes of gp96/GRP94 and peptides have been shown to elicit immunogenicity. We used fluorescence to understand peptide association with gp96. A pyrene-peptide conjugate was complexed with gp96 under a variety of conditions. At room temperature in low salt (20 mM NaCl), the peptide binds gp96 with a strong affinity (approximately 100-150 nM) and forms pyrene excimers, suggesting that the peptides were assembled as dimers. In high salt (2.2 M NaCl), although peptide binding was stronger (Ka approximately 55 nM) than in low salt, pyrene excimers were absent, implying that peptides were farther apart in the complex. Heat shock-activated peptide binding exhibited characteristics of both low salt and high salt modes of binding. Anisotropy and average lifetime of the bound pyrene suggested that peptides were probably located in a solvent-accessible hydrophobic binding pocket in low salt, whereas in high salt, the peptide may be buried in a less hydrophobic (more hydrophilic) environment. These results suggested that peptide-gp96 complexes were assembled in several different ways, depending on the assembly conditions. Resonance energy transfer between the intrinsic tryptophan(s) in gp96 and pyrene suggested that one or more tryptophan residues were within the critical Forster distance of 27-30 A from the pyrene in the bound peptide. It is proposed that peptides are assembled within higher order gp96 complexes (dimers, etc.) in a hydrophobic pocket and that there may be a conformational change in gp96 leading to an open configuration for peptide loading.     

Sequence dependence of kinetics and morphology of collagen model peptide self-assembly into higher order structures

The process of self-assembly of the triple-helical peptide (Pro-Hyp-Gly)(10) into higher order structure resembles the nucleation-growth mechanism of collagen fibril formation in many features, but the irregular morphology of the self-assembled peptide contrasts with the ordered fibers and networks formed by collagen in vivo. The amino acid sequence in the central region of the (Pro-Hyp-Gly)(10) peptide was varied and found to affect the kinetics of self-assembly and nature of the higher order structure formed. Single amino acid changes in the central triplet produced irregular higher order structures similar to (Pro-Hyp-Gly)(10), but the rate of self-association was markedly delayed by a single change in one Pro to Ala or Leu. The introduction of a Hyp-rich hydrophobic sequence from type IV collagen resulted in a more regular suprastructure of extended fibers that sometimes showed supercoiling and branching features similar to those seen for type IV collagen in the basement membrane network. Several peptides, where central Pro-Hyp sequences were replaced by charged residues or a nine-residue hydrophobic region from type III collagen, lost the ability to self-associate under standard conditions. The inability to self-assemble likely results from loss of imino acids, and lack of an appropriate distribution of hydrophobic/electrostatic residues. The effect of replacement of a single Gly residue was also examined, as a model for collagen diseases such as osteogenesis imperfecta and Alport syndrome. Unexpectedly, the Gly to Ala replacement interfered with self-assembly of (Pro-Hyp-Gly)(10), while the peptide with a Gly to Ser substitution self-associated to form a fibrillar structure.     

Predictive modeling of self assembly of chromonics materials

This study identifies mechanisms of self-assembly of a chromonic molecule with interesting self-assembly properties. Results from molecular dynamics (MD) studies used to understand the moieties of the molecule that affect the structure are consistent with experimental observations of these self-assembled structures. Coulombic forces, with significant contributions from π–π interactions drives the self-assembly of this class of materials; hydrogen bond energies are also significant.     

Molecular engineering of antimicrobial peptides: microbial targets,peptide motifs and translation opportunities

The global public health threat of antimicrobial resistance has led the scientific community to highly engage into research on alternative strategies to the traditional small molecule therapeutics. Here, we review one of the most popular alternatives amongst basic and applied research scientists, synthetic antimicrobial peptides. The ease of peptide chemical synthesis combined with emerging engineering principles and potent broad-spectrum activity, including against multidrug-resistant strains, has motivated intense scientific focus on these compounds for the past decade. This global effort has resulted in significant advances in our understanding of peptide antimicrobial activity at the molecular scale. Recent evidence of molecular targets other than the microbial lipid membrane, and efforts towards consensus antimicrobial peptide motifs, have supported the rise of molecular engineering approaches and design tools, including machine learning. Beyond molecular concepts, supramolecular chemistry has been lately added to the debate; and helped unravel the impact of peptide self-assembly on activity, including on biofilms and secondary targets, while providing new directions in pharmaceutical formulation through taking advantage of peptide self-assembled nanostructures. We argue that these basic research advances constitute a solid basis for promising industry translation of rationally designed synthetic peptide antimicrobials, not only as novel drugs against multidrug-resistant strains but also as components of emerging antimicrobial biomaterials. This perspective is supported by recent developments of innovative peptide-based and peptide-carrier nanobiomaterials that we also review.     

Molecular dynamics study of small PNA molecules in lipid-water system

We present the results of molecular dynamics simulations of small peptide nucleic acid (PNA) molecules, synthetic analogs of DNA, at a lipid bilayer in water. At neutral pH, without any salt, and in the NP(n)gammaT ensemble, two similar PNA molecules (6-mers) with the same nucleic base sequence and different terminal groups are investigated at the interface between water and a 1-palmitoyl-2-oleoylphosphatidylcholine lipid bilayer. The results of our simulations suggest that at low ionic strength of the solution, both PNA molecules adsorb at the lipid-water interface. In the case where the PNA molecule has charged terminal groups, the main driving force of adsorption is the electrostatic attraction between the charged groups of PNA and the lipid heads. The main driving force of adsorption of the PNA molecule with neutral terminal groups is the hydrophobic interaction of the nonpolar groups. Our simulations suggest that the system free energy change associated with PNA adsorption at the lipid-water interface is on the order of several tens of kT per PNA molecule in both cases.     

Tuning β-sheet peptide self-assembly and hydrogelation behavior by modification of sequence hydrophobicity and aromaticity

Peptide self-assembly leading to cross-β amyloid structures is a widely studied phenomenon because of its role in amyloid pathology and the exploitation of amyloid as a functional biomaterial. The self-assembly process is governed by hydrogen bonding, hydrophobic, aromatic π-π, and electrostatic Coulombic interactions. A role for aromatic π-π interactions in peptide self-assembly leading to amyloid has been proposed, but the relative contributions of π-π versus general hydrophobic interactions in these processes are poorly understood. The Ac-(XKXK)(2)-NH(2) peptide was used to study the contributions of aromatic and hydrophobic interactions to peptide self-assembly. Position X was globally replaced by valine (Val), isoleucine (Ile), phenylalanine (Phe), pentafluorophenylalanine (F(5)-Phe), and cyclohexylalanine (Cha). At low pH, these peptides remain monomeric because of repulsion of charged lysine (Lys) residues. Increasing the solvent ionic strength to shield repulsive charge-charge interactions between protonated Lys residues facilitated cross-β fibril formation. It was generally found that as peptide hydrophobicity increased, the required ionic strength to induce self-assembly decreased. At [NaCl] ranging from 0 to 1000 mM, the Val sequence failed to assemble. Assembly of the Phe sequence commenced at 700 mM NaCl and at 300 mM NaCl for the less hydrophobic Ile variant, even though it displayed a mixture of random coil and β-sheet secondary structures over all NaCl concentrations. β-Sheet formation for F(5)-Phe and Cha sequences was observed at only 20 and 60 mM NaCl, respectively. Whereas self-assembly propensity generally correlated to peptide hydrophobicity and not aromatic character the presence of aromatic amino acids imparted unique properties to fibrils derived from these peptides. Nonaromatic peptides formed fibrils of 3-15 nm in diameter, whereas aromatic peptides formed nanotape or nanoribbon architectures of 3-7 nm widths. In addition, all peptides formed fibrillar hydrogels at sufficient peptide concentrations, but nonaromatic peptides formed weak gels, whereas aromatic peptides formed rigid gels. These findings clarify the influence of aromatic amino acids on peptide self-assembly processes and illuminate design principles for the inclusion of aromatic amino acids in amyloid-derived biomaterials.     

The MHC class II molecule I-Ag7 exists in alternate conformations that are peptide dependent

Insulin-dependent diabetes mellitus is an autoimmune disease that is genetically linked to the HLA class II molecule DQ in humans and to MHC I-Ag7 in nonobese diabetic mice. The I-Ag7 beta-chain is unique and contains multiple polymorphisms, at least one of which is shared with DQ alleles linked to insulin-dependent diabetes mellitus. This polymorphism occurs at position 57 in the beta-chain, in which aspartic acid is mutated to a serine, a change that results in the loss of an interchain salt bridge between alphaArg76 and betaAsp57 at the periphery of the peptide binding groove. Using mAbs we have identified alternative conformations of I-Ag7 class II molecules. By using an invariant chain construct with various peptides engineered into the class II-associated invariant chain peptide (CLIP) region we have found that formation of these conformations is dependent on the peptide occupying the binding groove. Blocking studies with these Abs indicate that these conformations are present at the cell surface and are capable of interactions with TCRs that result in T cell activation.     

Synthesis and mass spectrometric characterization of a metal-affinity decapeptide: copper-induced conformational changes

A decapeptide with high affinity toward heavy metal ions (RCHQYHHNRE) has been prepared by Fmoc strategy using TGR resin as solid support. The model peptide provides a simple system that can be used for a systematic study of the impact of different metal ions on peptide secondary structure on a molecular level; histidine residues were incorporated into the peptide in a sequence similar to beta-amyloid peptide (Abeta1-40) to generate possible complexation sites for Cu (2+) ions. The peptide secondary structure, as investigated by circular dichroism, and self-assembled nanostructures were observed to depend strongly on the presence of copper and sodium dodecyl sulfate (SDS). Atomic force microscopy (AFM) revealed also that copper and SDS affected slightly the Abeta1-40 nanostructures. An explanation for the effect of metal ions and SDS on the self-assembly of peptides was proposed. The extensive beta-sheet formation may further promote peptide self-assembly into longer fibers.     

Effect of salt on the interactions of antimicrobial peptides with zwitterionic lipid bilayers

The effect of salt on the binding of the antimicrobial peptide magainin to POPC lipid bilayers is studied by 40-50 ns molecular dynamics simulations of a POPC bilayer in the presence of different concentrations of Na+ and Cl− ions, corresponding to effective concentrations of 0, 100, 150, 200, 250 and 300 millimolar NaCl, with and without a single molecule of antimicrobial peptide magainin. Simulations without magainin showed that increasing salt concentration leads to the decrease in the area per lipid, a decrease in the head group tilt of the lipids, as well as increased order of lipid tails, in agreement with other recent simulations. Simulations with magainin show that peptide binding to the lipids is stronger at lower concentrations of salt. The peptides disorder the lipids in their immediate vicinity, but this effect is diminished as the salt concentration increases. Our studies indicate that while 50 ns simulations give information on peptide hydrogen bonding and lipid tail ordering that is insensitive to the initial peptide orientation, this run time is not sufficient to equilibrate the peptide position and orientation within the bilayer.     

Effect of salt on the interactions of antimicrobial peptides with zwitterionic lipid bilayers

The effect of salt on the binding of the antimicrobial peptide magainin to POPC lipid bilayers is studied by 40-50 ns molecular dynamics simulations of a POPC bilayer in the presence of different concentrations of Na+ and Cl- ions, corresponding to effective concentrations of 0, 100, 150, 200, 250 and 300 millimolar NaCl, with and without a single molecule of antimicrobial peptide magainin. Simulations without magainin showed that increasing salt concentration leads to the decrease in the area per lipid, a decrease in the head group tilt of the lipids, as well as increased order of lipid tails, in agreement with other recent simulations. Simulations with magainin show that peptide binding to the lipids is stronger at lower concentrations of salt. The peptides disorder the lipids in their immediate vicinity, but this effect is diminished as the salt concentration increases. Our studies indicate that while 50 ns simulations give information on peptide hydrogen bonding and lipid tail ordering that is insensitive to the initial peptide orientation, this run time is not sufficient to equilibrate the peptide position and orientation within the bilayer.     

Effect of chirality of small molecule organofluorine inhibitors of amyloid self-assembly on inhibitor potency

The effect of enantiomeric trifluoromethyl-indolyl-acetic acid ethyl esters on the fibrillogenesis of Alzheimer’s amyloid β (Aβ) peptide is described. These compounds have been previously identified as effective inhibitors of the Aβ self-assembly in their racemic form. Thioflavin-T Fluorescence Spectroscopy and Atomic Force Microscopy were applied to assess the potency of the chiral target compounds. Both enantiomers showed significant inhibition in the in vitro assays. The potency of the enantiomeric inhibitors appeared to be very similar to each other suggesting the lack of the stereospecific binding interactions between these small molecule inhibitors and the Aβ peptide.