Comparisons of β-Hairpin Propensity Among Peptides with Homochiral or Heterochiral Strands

Assemblies of racemic β-sheet-forming peptides have attracted attention for biomedical applications because racemic forms of peptides can self-associate more avidly than do single enantiomers. In 1953, Pauling and Corey proposed “rippled β-sheet” modes of H-bond-mediated interstrand assembly for alternating L- and D-peptide strands; this structural hypothesis was complementary to their proposal of “pleated β-sheet” assembly for L-peptides. Although no high-resolution structure has been reported for a rippled β-sheet, there is strong evidence for the occurrence of rippled β-sheets in some racemic peptide assemblies. Here we compare propensities of peptide diastereomers in aqueous solution to form a minimum increment of β-sheet in which two antiparallel strands associate. β-Hairpin folding is observed for homochiral peptides with aligned nonpolar side chains, but no β-hairpin population can be detected for diastereomers in which one strand contains L residues and the other contains D residues. These observations suggest that rippled β-sheet assemblies are stabilized by interactions between β-sheet layers rather than interactions within these layers.     

A Small Cyclic β-Hairpin Peptide Mimics the Rbfox2 RNA Recognition Motif and Binds to the Precursor miRNA 20b

The RNA recognition motif (RRM), which is the most abundant RNA-binding motif in eukaryotes, is a well-structured domain of about 90 amino acids, yet the β2β3 hairpin, corresponding to strands 2 and 3 of the β-sheet, and the intervening loop make essential interactions with RNA in many RRM complexes. A series of small cyclic peptide mimics of the β2β3 hairpin of Rbfox2 protein that recognize the terminal loop of precursor miR-20b have been designed to investigate whether the full RNA-binding protein can be mimicked with a minimal structurally preorganized peptide. Within a small library of seven cyclic peptides, a peptide with low-micromolar affinity for the miR-20b precursor was found. NMR spectroscopy titration data suggest that this peptide specifically targets the apical loop of pre-miR-20b. This work shows that it is possible to mimic RNA-binding proteins with designed stable peptides, which provide a starting point for designing or evolving small peptide mimetics of RRM proteins.     

A Conformationally Stable Acyclic β-Hairpin Scaffold Tolerating the Incorporation of Poorly β-Sheet-Prone Amino Acids

The β-hairpin is a structural element of native proteins, but it is also a useful artificial scaffold for finding lead compounds to convert into peptidomimetics or non-peptide structures for drug discovery. Since linear peptides are synthetically more easily accessible than cyclic ones, but are structurally less well-defined, we propose XWXWXpPXK(/R)X(R) as an acyclic but still rigid β-hairpin scaffold that is robust enough to accommodate different types of side chains, regardless of the secondary-structure propensity of the X residues. The high conformational stability of the scaffold results from tight contacts between cross-strand cationic and aromatic side chains, combined with the strong tendency of the d -Pro-l -Pro dipeptide to induce a type II′ β-turn. To demonstrate the robustness of the scaffold, we elucidated the NMR structures and performed molecular dynamics (MD) simulations of a series of peptides displaying mainly non-β-branched, poorly β-sheet-prone residues at the X positions. Both the NMR and MD data confirm that our acyclic β-hairpin scaffold is highly versatile as regards the amino-acid composition of the β-sheet face opposite to the cationic−aromatic one.     

Modular Design of Supramolecular Ionic Peptides with Cell-Selective Membrane Activity

The rational design of materials with cell-selective membrane activity is an effective strategy for the development of targeted molecular imaging and therapy. Here we report a new class of cationic multidomain peptides (MDPs) that can undergo enzyme-mediated molecular transformation followed by supramolecular assembly to form nanofibers in which cationic clusters are presented on a rigid β-sheet backbone. This structural transformation, which is induced by cells overexpressing the specific enzymes, led to a shift in the membrane perturbation potential of the MDPs, and consequently enhanced cell uptake and drug delivery efficacy. We envision the directed self-assembly based on modularly designed MDPs as a highly promising approach to generate dynamic supramolecular nanomaterials with emerging membrane activity for a range of disease targeted molecular imaging and therapy applications.     

The Toxicity of Amyloid β Oligomers

In this review, we elucidate the mechanisms of Aβ oligomer toxicity which may contribute to Alzheimer's disease (AD). In particular, we discuss on the interaction of Aβ oligomers with the membrane through the process of adsorption and insertion. Such interaction gives rises to phase transitions in the sub-structures of the Aβ peptide from α-helical to β-sheet structure. By means of a coarse-grained model, we exhibit the tendency of β-sheet structures to aggregate, thus providing further insights to the process of membrane induced aggregation. We show that the aggregated oligomer causes membrane invagination, which is a precursor to the formation of pore structures and ion channels. Other pathological progressions to AD due to Aβ oligomers are also covered, such as their interaction with the membrane receptors, and their direct versus indirect effects on oxidative stress and intraneuronal accumulation. We further illustrate that the molecule curcumin is a potential Aβ toxicity inhibitor as a β-sheet breaker by having a high propensity to interact with certain Aβ residues without binding to them. The comprehensive understanding gained from these current researches on the various toxicity mechanisms show promises in the provision of better therapeutics and treatment strategies in the near future.     

Bacterial inclusion bodies of Alzheimer's disease β-amyloid peptides can be employed to study native-like aggregation intermediate states

The structures of oligomeric intermediate states in the aggregation process of Alzheimer's disease β-amyloid peptides have been the subject of debate for many years. Bacterial inclusion bodies contain large amounts of small heat shock proteins (sHSPs), which are highly homologous to those found in the plaques of the brains of Alzheimer's disease patients. sHSPs break down amyloid fibril structure in vitro and induce oligomeric assemblies. Prokaryotic protein overexpression thus mimics the conditions encountered in the cell under stress and allows the structures of Aβ aggregation intermediate states to be investigated under native-like conditions, which is not otherwise technically possible. We show that IB40/IB42 fulfil all the requirements to be classified as amyloids: they seed fibril growth, are Congo red positive and show characteristic β-sheet-rich CD spectra. However, IB40 and IB42 are much less stable than fibrils formed in vitro and contain significant amounts of non-β-sheet regions, as seen from FTIR studies. Quantitative analyses of solution-state NMR H/D exchange rates show that the hydrophobic cores involving residues V18-F19-F20 adopt β-sheet conformations, whereas the C termini adopt α-helical coiled-coil structures. In the past, an α-helical intermediate-state structure has been postulated, but could not be verified experimentally. In agreement with the current literature, in which Aβ oligomers are described as the most toxic state of the peptides, we find that IB42 contains SDS-resistant oligomers that are more neurotoxic than Aβ42 fibrils. E. coli inclusion bodies formed by the Alzheimer's disease β-amyloid peptides Aβ40 and Aβ42 thus behave structurally like amyloid aggregation intermediate states and open the possibility of studying amyloids in a native-like, cellular environment.     

Conformational Analysis of Polypeptides and Proteins for the Study of Protein Folding,Molecular Recognition,and Molecular Design

Conformational energy calculations provide an understanding as to how interatomic interactions lead to the three-dimensional structures of polypeptides and proteins, and how these molecules interact with other molecules. Illustrative results of such calculations pertain to model systems (α-helices and β-sheets, and interactions between them), to various open-chain and cyclic peptides, to fibrous proteins, to globular proteins, and to enzyme-substrate complexes. In most cases, the validity of the computations is established by experimental tests of the predicted structures.     

Effects of Arginine Deimination and Citrulline Side-Chain Length on Peptide Secondary Structure Formation

Post-translational modifications expand the chemical functionality of peptides and proteins beyond that originating from the encoded amino acids, but studies on the structural effects of these modifications have been limited. Arginine undergoes deimination to give citrulline (Cit), converting the positively charged guanidinium moiety into a neutral urea group. Herein, we report the effect of Arg deimination on secondary structure formation. To understand the reason for the number of methylene units in Cit, the effect of Cit side-chain length on secondary structure formation was also studied. Ala-based peptides and β-hairpin peptides were used to study α-helix and β-sheet formation, respectively. Peptides containing Cit analogues were prepared by an orthogonal protecting group strategy coupled with solid-phase carbamylation. The CD data for the Ala-based peptides were analyzed by using modified Lifson–Roig theory, showing that the helix propensity of Arg decreased upon deimination and that either shortening or lengthening Cit also decreased the helix propensity. The β-hairpin peptides were analyzed by NMR methods, showing minimal change in strand formation energetics upon Arg deimination. Altering the Cit side-chain length did not affect strand formation energetics either. These results should be useful for the preparation of urea-bearing systems and the design of peptides incorporating urea-bearing residues with varying side-chain length.     

Hydrogen Bond Surrogate-Constrained Dynamic Antiparallel β-Sheets

Antiparallel β-sheets are important secondary structures within proteins that equilibrate with random-coil states; however, little is known about the exact dynamics of this process. Here, the first dynamic β-sheet models that mimic this equilibrium have been designed by using an H-bond surrogate that introduces constraint and torque into a tertiary amide bond. 2D NMR data sufficiently reveal the structure, kinetics, and thermodynamics of the folding process, thereby leading the way to similar analysis in isolated biologically relevant β-sheets.     

Deep Sequencing of a Systematic Peptide Library Reveals Conformationally-Constrained Protein Interface Peptides that Disrupt a Protein-Protein Interaction

Disrupting protein-protein interactions is difficult due to the large and flat interaction surfaces of the binding partners. The BLIP and BLIP-II proteins are unrelated in sequence and structure and yet each potently inhibit β-lactamases. High-throughput oligonucleotide synthesis was used to construct a 12,470-member library containing overlapping linear and cyclic peptides ranging in size from 6 to 21 amino acids that scan through the sequences of BLIP and BLIP-II. Phage display affinity selections and deep sequencing revealed that, despite the differences in interaction surfaces with β-lactamases, rapid enrichment of consensus peptide regions originating from both BLIP and BLIP-II contact residues in the binding interface occurred. BLIP and BLIP-II peptides that were enriched by affinity selection were shown to bind β-lactamases and disrupt the BLIP/β-lactamase interaction. The results suggest that peptides that bind at and disrupt PPI interfaces can be identified through systematic peptide library construction, affinity selection, and deep sequencing.     

Polymer molecular behaviors at buried polymer/metal and polymer/polymer interfaces and their relations to adhesion in packaging

Packaging materials are widely used in modern microelectronics. The interfacial structures of packaging materials determine the adhesion properties of these materials. Weak adhesion or delamination at interfaces involving packaging materials can lead to failure of microelectronic devices. Therefore, it is important to investigate the molecular structures of such interfaces. However, it is difficult to study molecular structures of buried interfaces due to the lack of appropriate analytical techniques. Sum frequency generation (SFG) vibrational spectroscopy has recently been used to probe buried solid/solid interfaces to understand molecular structures and behaviors such as the presence, coverage, ordering, orientation, and diffusion of functional groups at buried interfaces and their relations to adhesion in situ in real time. In this review, we describe our recent progress in the development of nondestructive methodology to examine buried polymer/metal interfaces and summarize how the developed methodology has been used to elucidate adhesion mechanisms at buried polymer/metal interfaces using SFG. We also elucidated the molecular interactions between polymers and various model and commercial epoxy materials, and the correlations between such interactions and the interfacial adhesion, providing in-depth understanding on the adhesion mechanisms of polymer adhesives.     

Chiral vibrational structures of proteins at interfaces probed by sum frequency generation spectroscopy

We review the recent development of chiral sum frequency generation (SFG) spectroscopy and its applications to study chiral vibrational structures at interfaces. This review summarizes observations of chiral SFG signals from various molecular systems and describes the molecular origins of chiral SFG response. It focuses on the chiral vibrational structures of proteins and presents the chiral SFG spectra of proteins at interfaces in the C-H stretch, amide I, and N-H stretch regions. In particular, a combination of chiral amide I and N-H stretches of the peptide backbone provides highly characteristic vibrational signatures, unique to various secondary structures, which demonstrate the capacity of chiral SFG spectroscopy to distinguish protein secondary structures at interfaces. On the basis of these recent developments, we further discuss the advantages of chiral SFG spectroscopy and its potential application in various fields of science and technology. We conclude that chiral SFG spectroscopy can be a new approach to probe chiral vibrational structures of protein at interfaces, providing structural and dynamic information to study in situ and in real time protein structures and dynamics at interfaces.     

Understanding surfaces and buried interfaces of polymer materials at the molecular level using sum frequency generation vibrational spectroscopy

This paper reviews recent progress in the studies on polymer surfaces/interfaces using sum frequency generation (SFG) vibrational spectroscopy. SFG theory, technique, and some experimental details have been presented. The review is focused on the SFG studies on buried interfaces involving polymer materials, such as polymer–water interfaces and polymer–polymer interfaces. Molecular interactions between polymer surfaces and adhesion promoters as well as biological molecules such as proteins and peptides have also been elucidated using SFG. This review demonstrates that SFG is a powerful technique to characterize molecular level structural information of complicated polymer surfaces and interfaces in situ. Copyright © 2006 Society of Chemical Industry     

Towards Supramolecular Catalysis with Small Self-assembled Peptides

Self-assembly of small peptides offers unique opportunities for the bottom-up construction of supramolecular catalysts that aim to emulate the efficiency and selectivity of natural enzymes. Small, information-rich, simple molecules based on amino acids can self-organise autonomously into complex systems with emergent catalytic properties. The power of noncovalent interactions can be used to construct supramolecular peptidic tertiary structures. Moreover, specific functional groups present in amino acid side-chains may present either a catalytic activity by themselves or be able to bind cofactors such as metal ions. In this scenario, although relevant progress has been achieved in recent years, promising applications in biomaterials science are foreseen. In this review, we discuss the state-of-the-art of this approach at the interface between supramolecular chemistry and peptide science.     

Design and application of stimulus-responsive peptide systems

The ability of peptides and proteins to change conformations in response to external stimuli such as temperature, pH and the presence of specific small molecules is ubiquitous in nature. Exploiting this phenomenon, numerous natural and designed peptides have been used to engineer stimulus-responsive systems with potential applications in important research areas such as biomaterials, nanodevices, biosensors, bioseparations, tissue engineering and drug delivery. This review describes prominent examples of both natural and designed synthetic stimulus-responsive peptide systems. While the future looks bright for stimulus-responsive systems based on natural and rationally engineered peptides, it is expected that the range of stimulants used to manipulate such systems will be significantly broadened through the use of combinatorial protein engineering approaches such as directed evolution. These new proteins and peptides will continue to be employed in exciting and high-impact research areas including bionanotechnology and synthetic biology.     

Interaction of self-assembling β-sheet peptides with phospholipid monolayers: The effect of serine, threonine, glutamine and asparagine amino acid side chains

The dioleoyl phosphatidylcholine (DOPC) monolayer activities of 11 systematically altered 11 residue β-sheet tape-forming peptides were studied. Peptide-DOPC interactions were characterised by electrochemical impedance spectroscopy (EIS). An impedance model combining the constant phase element approach with dielectric relaxation in the surface layer was used to analyse the data. The facilitation of DOPC layer permeability to ions by the peptides was monitored by both EIS and the Tl(I)/Tl(Hg) and Cd(II)/Cd(Hg) faradaic reactions. It was found that peptides with side chains of serine and threonine interact with DOPC layers more strongly and in a well characterised manner compared to peptides with side chains of glutamine and asparagine. Cationic and neutral peptides containing serine and threonine penetrate the DOPC to give a maximum plateau monolayer capacitance. At higher solution concentrations of these peptides the growth of a well-defined secondary element in the impedance data indicates the segregation of secondary DOPC-peptide phases. Cationic and neutral peptides containing serine and anionic peptides containing threonine interact with the DOPC layers leading to a selective increase in the layer's permeability to Tl⁺ ions. Impedance measurements at higher solution concentrations of anionic peptides with serine and threonine show that these peptide modified DOPC layers associate with electrolyte ions.     

Understanding the Interactions of Guanine Quadruplexes with Peptides as Novel Strategies for Diagnosis or Tuning Biological Functions

Guanine quadruplexes (G4s) are nucleic acid structures exhibiting a complex structural behavior and exerting crucial biological functions in both cells and viruses. The specific interactions of peptides with G4s, as well as an understanding of the factors driving the specific recognition are important for the rational design of both therapeutic and diagnostic agents. In this review, we examine the most important studies dealing with the interactions between G4s and peptides, highlighting the strengths and limitations of current analytic approaches. We also show how the combined use of high-level molecular simulation techniques and experimental spectroscopy is the best avenue to design specifically tuned and selective peptides, thus leading to the control of important biological functions.     

An Engineered β-Hairpin Peptide Forming Thermostable Complexes with ZnII,NiII, and CuII through a His3 Site

The three-dimensional structure of a peptide, which determines its function, can denature at elevated temperatures, in the presence of chaotropic reagents, or in organic solvents. These factors limit the applicability of peptides. Herein, we present an engineered β-hairpin peptide containing a His₃ site that forms complexes with Zn^II, Ni^II, and Cu^II. Circular dichroism spectroscopy shows that the peptide−metal complexes exhibit melting temperatures up to 80 °C and remain folded in 6 M guanidine hydrochloride as well as in organic solvents. Intrinsic fluorescence titration experiments were used to determine the dissociation constants of metal binding in the nano- to sub-nanomolar range. The coordination geometry of the peptide−Cu^II complex was studied by EPR spectroscopy, and a distorted square planar coordination geometry with weak interactions to axial ligands was revealed. Due to their impressive stability, the presented peptide−metal complexes open up interesting fields of application, such as the development of a new class of peptide−metal catalysts for stereoselective organic synthesis or the directed design of extremophilic β-sheet peptides.     

Cationic Amphiphilic Peptides: Synthetic Antimicrobial Agents Inspired by Nature

Antimicrobial peptides are ubiquitous in multicellular organisms and have served as defense mechanisms for their successful evolution and throughout their life cycle. These peptides are short cationic amphiphilic polypeptides of fewer than 50 amino acids containing either a few disulfide-linked cysteine residues with a characteristic β-sheet-rich structure or linear α-helical conformations with hydrophilic side chains at one side of the helix and hydrophobic side chains on the other side. Antimicrobial peptides cause bacterial cell lysis either by direct cell-surface damage via electrostatic interactions between the cationic side chains of the peptide and the negatively charged cell surface, or by indirect modulation of the host defense systems. Electrostatic interactions lead to bacterial cell membrane disruption followed by leakage of cellular components and finally bacterial cell death. Because of their unusual mechanism of cell damage, antimicrobial peptides are effective against drug-resistant bacteria and may therefore prove more effective than classical antibiotics in certain cases. Currently, around 3000 natural antimicrobial peptides from six kingdoms (bacteria, archaea, protists, fungi, plants, and animals) have been isolated and sequenced. However, only a few of them are under clinical trials and/or in the commercial development stage for the treatment of bacterial infections caused by antibiotic-resistant bacteria. Moreover, high structural complexity, poor pharmacokinetic properties, and low antibacterial activity of natural antimicrobial peptides hinder their progress in drug development. To overcome these hurdles, researchers have become increasingly interested in modification and nature-inspired synthetic antimicrobial peptides. This review discusses some of the recent studies reported on antimicrobial peptides.     

Investigation on the Interactions between Self-Assembled β-Sheet Peptide Nanofibers and Model Cell Membranes

Self-assembled peptide nanofibers (NFs) obtained from β-sheet peptides conjugated with drugs, including antigenic peptides, have recently attracted significant attention. However, extensive studies on the interactions of β-sheet peptide NFs with model cell membranes have not been reported. In this study, we investigated the interactions between three types of NFs, composed of PEG-peptide conjugates with different ethylene glycol (EG) lengths (6-, 12- and 24-mer), and dipalmitoylphosphatidylcholine (DPPC) Langmuir membranes. When increasing the EG chain length, those interactions significantly decreased considering measurements in the presence of the NFs of: (i) changes in surface pressure of the DPPC Langmuir monolayers and (ii) surface pressure–area (π–A) compression isotherms of DPPC. Because the observed trend was similar to the EG length dependency with regard to cellular association and cytotoxicity of the NFs that was reported previously, the interaction of NFs with phospholipid membranes represented a crucial factor to determine the cellular association and toxicity of the NFs. In contrast to NFs, no changes were observed with varying EG chain length on the interaction of the building block peptide with the DPPC membrane. The results obtained herein can provide a design guideline on the formulation of β-sheet peptide NFs, which may broaden its potential.