Understanding self-assembled amphiphilic peptide supramolecular structures from primary structure helix propensity

Small amphiphilic peptides are attractive building blocks to design biocompatible supramolecular structures via self-assembly, with applications in, for example, drug delivery, tissue engineering, and nanotemplating. We address the influence of systematical changes in the amino acid sequence of such peptides on the self-assembled macromolecular structures. For cationic-head surfactant-like eight-residue peptides, the apolar tail amino acids were chosen to systematically vary the propensity to form an alpha-helical secondary structure while conserving the overall hydrophobicity of the sequence. Characterization of the supramolecular structures indicates that for short peptides a beta-sheet secondary structure correlates with ribbonlike assemblies while random-coil and alpha-helical secondary structures correlate with assembly of rods.     

Self-assembly of surfactant-like peptides and their applications

Numerous peptides derived from naturally occurring proteins or de novo designed have been found to self-assemble into various nanostructures.These well-defined nanostructures have shown great potential for a variety of biomedical and biotechnological applications.In particular,surfactant-like peptides(SLPs)have distinctive advantages in their length,aggregating ability,and water solubility.In this article,we report recent advances in the mechanistic understanding of the self-assembly principles of SLPs and in their applications,most of which have been made in our laboratory.Hydrogen bonding between peptide backbones,hydrophobic interaction between hydrophobic side chains,and electrostatic repulsion between charged head groups all have roles in mediating the self-assembly of SLPs;the final self-assembled nanostructures are therefore dependent on their interplay.SLPs have shown diverse applications ranging from membrane protein stabilization and antimicrobial/anticancer agents to nanofabrication and biomineralization.Future advances in the self-assembly of SLPs will hinge on their large-scale production,the design of new functional SLPs with targeted properties,and the exploitation of new or improved applications.     

Templated growth of hybrid structures at the peptide-peptide interface

This study describes the use of peptide vesicular platforms for the templated growth of fibrillar structures to craft hybrids that retain the gross morphological features of two discreet self-assembled peptides. A synthetic triskelion peptide, which results in the rapid emergence of self-assembled spherical structures, was employed as a template. Addition of either one of two different peptides, both of which form long filamentous structures when co-incubated with the triskelion solution, affords hybrids that retain the gross morphology of both the spherical and filamentous structures. It is surmised that this process is aided by hydrogen bonding and the interdigitation of aromatic residues, which leads to the growth of hybrid structures. We believe that observations concerning the surface-assisted growth of peptide fibrils and tubular structures from vesicular platforms may have ramifications for the design and development of peptide-based hybrid materials with controlled hierarchical structures.     

Self-assembly of short amyloidogenic peptides at the air-water interface

Short peptide stretches in amyloidogenic proteins can form amyloid fibrils in vitro and have served as good models for studying amyloid fibril formation. Recently, these amyloidogenic peptides have gained considerable attention, as non-amyloid ordered structures can be obtained from these peptides by carefully tuning the conditions of self-assembly, especially pH, temperature and presence of organic solvents. We have examined the effect of surface pressure on the self-assembled structures of two amyloidogenic peptides, Pβ(2)m (Ac-DWSFYLLYYTEFT-am) and AcPHF6 (Ac-VQIVYK-am) at the air-water interface when deposited from different solvents. Both the peptides are surface-active and form Thioflavin T (ThT) positive structures at the air-water interface. There is considerable hysteresis in the compression and expansion isotherms, suggesting the occurrence of structural rearrangements during compression. Preformed Pβ(2)m fibrillar structures at the air-water interface are disrupted as peptide is compressed to lower molecular areas but restored if the film is expanded, suggesting that the process is reversible. AcPHF6, on the other hand, shows largely sheet-like structures at lower molecular areas. The solvents used for dissolution of the peptides appear to influence the nature of the aggregates formed. Our results show that like hydrostatic pressure, surface pressure can also be utilized for modulating the self-assembly of the amyloidogenic and self-assembling peptides.     

Chiral Fibers Formation Upon Assembly of Tetraphenylalanine Peptide Conjugated to a PNA Dimer

Self-assembly of biomolecules such as peptides, nucleic acids or their analogues affords supramolecular objects, exhibiting structures and physical properties dependent on the amino-acid or nucleobase composition. Conjugation of the peptide diphenylalanine (FF) to peptide nucleic acids triggers formation of self-assembled structures, mainly stabilized by interactions between FF. In this work we report formation of homogeneous chiral fibers upon self-assembly of the hybrid composed of the tetraphenylalanine peptide (4F) conjugated to the PNA dimer adenine-thymine (at). In this case nucleobases seem to play a key role in determining the morphology and chirality of the fibers. When the PNA “at” is replaced by guanine-cytosine dimer “gc”, disordered structures are observed. Spectroscopic characterization of the self-assembled hybrids, along with AFM and SEM studies is reported. Finally, a structural model consistent with the experimental evidence has also been obtained, showing how the building blocks of 4Fat arrange to give helical fibers.     

Design of self-assembling surfactant-like peptides and their applications

A surfactant is briefly defined as a material that can greatly reduce the surface tension of water when used in very low concentrations. Surfactants are usually organic compounds that are amphiphilic, containing both hydrophobic groups and hydrophilic groups. Therefore, they are soluble in both organic solvents and water. Many surfactants can also assemble in the bulk solution into aggregates such as vesicles and micelles. Self-assembling peptides are a novel category of designer peptides that can undergo spontaneous organization into well-ordered nanostructures with the great potentials in nanotechnology, nanomedicine including 3-dimensional (3-D) cell culture, drug delivery, wound repair, and so on. In this review, we introduce a family of designer surfactant-like peptides: the self-assembling peptides which have been derived by mimicking the structure of traditional surfactants.     

Quantification of peptide components in cinobufacini injection and toad skin by ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry

Cinobufacini injection is commonly used in the clinical treatment of tumors and hepatitis B, but the quality is uneven. Currently, the main focus of its quality assessment is on steroids and alkaloids. Based on a previous study, we screened four peptides with high reproducibility, responsiveness, and specificity. This research was the first to develop an ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry approach for evaluating the quality of cinobufacini preparations from the peptide perspective. In this study, we have identified 230 peptides in cinobufacini injection by Q-Exactive mass spectrometry, which contains species-specific peptides. Then, we used ultra-high-performance liquid chromatography/triple quadrupole mass spectrometry to establish a quantitative method for species-specific peptides and carried out method validation. The result revealed that four peptides were linear in a specific range, and had great reproducibility, accuracy, and stability. Eventually, we evaluated the quality of eight batches of cinobufacini injections and 26 batches of toad skins using the total content of target peptides as the criterion. The outcomes demonstrated that the quality of cinobufacini injection is generally stable and the toad skin from Shandong is of the best quality. In conclusion, the quantitative approach that focuses on peptides will offer innovative perspectives on assessing the quality of cinobufacini preparations.     

The impact of impurities in synthetic peptides on the outcome of T-cell stimulation assays

Protein-spanning peptide pools have proven valuable as a screening tool for detecting T-lymphocyte responses against a wide range of proteins. We have used this approach in our search for T cells reactive to the onconeural protein HuD. We found positive responses in only 3 of 127 individuals; however, these were highly unusual in that the same class I HLA alleles and peptides were involved. These T-cell responses were not confirmed when peptides re-synthesized by the same manufacturer with similar and with higher purity levels were used. Our observations indicated that these T-cell responses were not directed against the designed HuD peptides. Here, we report on (i) comparisons of the peptide batches analyzed by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) that did--and did not--elicit T-cell responses and (ii) a detailed analysis of the various by-products of peptides, irrespective of T-cell assay outcome. We found numerous differences between the peptide batches, such as omissions of amino acids in the primary structure of the peptides. Furthermore, some batches revealed strong interactions with calcium ions or contained sulfated peptides. Our data reveal that different batches from the same peptide may contain artefacts that influence the outcome of HLA-restricted T-cell response assays.     

Recent advances in short peptide self-assembly: from rational design to novel applications

Owing to their structural simplicity and robust self-assembled nanostructures, short peptides prove to be an ideal system to explore the physical processes of self-assembly, hydrogels, semi-flexible polymers, quenched disorder, and reptation. Rational design in peptide sequences facilitates cost-effective manufacturing, but the huge number of possible peptides has imposed obstacles for their characterization to establish functional connections to the primary, secondary, and tertiary structures. This review aims to cover recent advances in the self-assembly of designed short peptides, with a focus on physical driving forces, design rules, characterization methods, and exemplar applications. Super-resolution microscopy in combination with modern image analysis have been applied to quantify the structure and dynamics of peptide hydrogels, while small-angle neutron scattering and solid-state nuclear magnetic resonance continue to provide valuable information on structures over complementary length scales. Short peptides are attractive in biomedicine and nanotechnology, e.g., as antimicrobials, anticancer agents, vehicles for controlled drug release, peptide bioelectronics, and responsive cell culture materials.     

Peptide Amphiphile Mediated Co-assembly for Nanoplasmonic Sensing

Aromatic interactions are commonly involved in the assembly of naturally occurring building blocks, and these interactions can be replicated in an artificial setting to produce functional materials. Here we describe a colorimetric biosensor using co-assembly experiments with plasmonic gold and surfactant-like peptides (SLPs) spanning a wide range of aromatic residues, polar stretches, and interfacial affinities. The SLPs programmed in DDD−(ZZ)_x−FFPC self-assemble into higher-order structures in response to a protease and subsequently modulate the colloidal dispersity of gold leading to a colorimetric readout. Results show the strong aggregation propensity of the FFPC tail without polar DDD head. The SLPs were specific to the target protease, i.e., M^pro, a biomarker for SARS-CoV-2. This system is a simple and visual tool that senses M^pro in phosphate buffer, exhaled breath condensate, and saliva with detection limits of 15.7, 20.8, and 26.1 nM, respectively. These results may have value in designing other protease testing methods.     

Morphology transformation via pH-triggered self-assembly of peptides

Three flexible peptides (P1: (C(17)H(35)CO-NH-GRGDG)(2)KG; P2: (Fmoc-GRGDG)(2)KG; P3: (CH(3)CO-NH-GRGDG)(2)KG) self-assembled to form a variety of morphologically distinct assemblies at different pHs. P1 formed nanofibers at pH 3, then self-assembled into nanospheres with pH up to 6 and further changed to lamellar structures when the pH value was further increased to 10. P2 aggregated into an entwined network structure at pH 3, and then self-assembled into well-defined nanospheres, lamellar structures, and vesicles via adjusting the pH value. However, P3 did not self-assemble into well-ordered nanostructures, presumably due to the absence of a large hydrophobic group. The varying self-assembly behaviors of the peptides at different pHs are attributed to molecular conformational changes. These self-assembled supramolecular materials might contribute to the development of new peptide-based biomaterials.     

Self-assembly of peptide scaffolds in biosilica formation: computer simulations of a coarse-grained model

The self-assembly of model peptides is studied using Brownian dynamics computer simulations. A coarse-grained, bead-spring model is designed to mimic silaffins, small peptides implicated in the biomineralization of certain silica diatom skeletons and observed to promote the formation of amorphous silica nanospheres in vitro. The primary characteristics of the silaffin are a 15 amino acid hydrophilic backbone and two modified lysine residues near the ends of the backbone carrying long polyamine chains. In the simulations, the model peptides self-assemble to form spherical clusters, networks of strands, or bicontinuous structures, depending on the peptide concentration and effective temperature. The results indicate that over a broad range of volume fractions (0.05-25%) the characteristic structural lengthscales fall in the range 12-45 nm. On this basis, we suggest that self-assembled structures act as either nucleation points or scaffolds for the deposition of 10-100 nm silica-peptide building blocks from which diatom skeletons and synthetic nanospheres are constructed.     

Solid-state NMR structural studies of peptides immobilized on gold nanoparticles

In this paper we describe solid-state NMR experiments that provide information on the structures of surface-immobilized peptides. The peptides are covalently bound to alkanethiolates that are self-assembled as monolayers on colloidal gold nanoparticles. The secondary structure of the immobilized peptides was characterized by quantifying the Ramachandran angles phi and psi. These angles were determined in turn from distances between backbone carbonyl 13C spins, measured with the double-quantum filtered dipolar recoupling with a windowless sequence experiment, and by determination of the mutual orientation of chemical shift anisotropy tensors of 13C carbonyl spins on adjacent peptide planes, obtained from the double-quantum cross-polarization magic-angle spinning spectrum. It was found that peptides composed of periodic sequences of leucines and lysines were bound along the length of the peptide sequence and displayed a tight alpha-helical secondary structure on the gold nanoparticles. These results are compared to similar studies of peptides immobilized on hydrophobic surfaces.     

Simple generalized reaction conditions for the conversion of primary aliphatic amines to surfactant-like guanidine salts with 1H-pyrazole carboxamidine hydrochloride

Improved reaction conditions for the electrophilic reaction between a free aliphatic amine and 1H-pyrazole carboxamidine have been discovered. The surfactant-like guanidine salts, which are often hard to work with, were obtained in decent yields with short reaction times, minimal workup, and high level of purity.     

Heterotypic Supramolecular Hydrogels Formed by Noncovalent Interactions in Inflammasomes

The advance of structural biology has revealed numerous noncovalent interactions between peptide sequences in protein structures, but such information is less explored for developing peptide materials. Here we report the formation of heterotypic peptide hydrogels by the two binding motifs revealed by the structures of an inflammasome. Specifically, conjugating a self-assembling motif to the positively or negatively charged peptide sequence from the ASCPYD filaments of inflammasome produces the solutions of the peptides. The addition of the peptides of the oppositely charged and complementary peptides to the corresponding peptide solution produces the heterotypic hydrogels. Rheology measurement shows that ratios of the complementary peptides affect the viscoelasticity of the resulted hydrogel. Circular dichroism indicates that the addition of the complementary peptides results in electrostatic interactions that modulate self-assembly. Transmission electron microscopy reveals that the ratio of the complementary peptides controls the morphology of the heterotypic peptide assemblies. This work illustrates a rational, biomimetic approach that uses the structural information from the protein data base (PDB) for developing heterotypic peptide materials via self-assembly.     

From fibers to micelles using point-mutated amphiphilic peptides

Biocompatible, self-assembled nanostructures are attracting ever more attention, in particular in aqueous media for biomedical applications. Here, we present the successful, solid-phase peptide synthesis (SPPS) and characterization of short amino acid sequences with amphiphilic character with the aim of gaining insight into their self-assembled, supramolecular structures. The peptide design includes three parts: (a) a charged lysine part, (b) an acetylated lysine part, and (c) a constant hydrophobic rodlike helix, based on gramicidin A (gA). By stepwise replacement of free lysine (K) with acetylated lysine (X) we generated a library of a total of 10 peptides, Ac-X(8)-gA and K(m)X(8-m)-gA (m ranging from 0 to 8). By using point mutations, we adjusted the degree of acetylation (DA) and thus the overall amphiphilicity of the peptides, which led to a change in the secondary structure in the aqueous environment from a β-sheet to an α-helix. This transition generated a significant change in the morphology of the self-assembled structures from fibers to micelles. Two different regions were observed for the conformation of the hydrophilic part of the peptide: one region, a β-sheet-like secondary structure, inducing fiber formation (high DA), the other an α-helical-like secondary structure, generating micelle formation (moderate and low DA). The micellar structures depended on the degree of acetylation, which influenced their critical micelle concentration (cmc). These morphology regions were determined by a combination of circular dichroism, dynamic light scattering, surface tension, and transmission electron microscopy, which allowed us to correlate the generated supramolecular architectures with the fine changes obtained by means of the point mutation strategy.     

Phage-chips for novel optically readable tissue engineering assays

We report novel phage-based array chips that are optically readable for cell proliferation and morphology assays. Using M13 phages that were engineered to display RGD on its major coat proteins and/or immobilize FGFb on its minor coat proteins, we prepared arrays of phage spot matrices composed of self-assembled nanofibrous network structures. We cultured fibroblasts on the arrays and, using surface plasmon resonance (SPR) spectroscopy, monitored the effects of the biochemical cues displayed by the phage on cell proliferation and morphology. This study demonstrates the utility of engineered phages as promising coating materials for lab-on-a-chip (LOC) platforms, allowing sensitive monitoring of the effects of functional peptides on cell growth. Phage-chips have great potential for use as high-throughput screening systems for biochemical assays and biosensors and the discovery of novel drugs.     

Evaporative self-assembly from complex DNA-colloid suspensions

Evaporation-induced pattern formation has attracted considerable attention as a simple yet versatile method for generating self-assembled structures that have broad applications from photonic devices to biomacromolecular recognition. Previous study of evaporative self-assembly has mainly focused on single nonvolatile component systems, and the driving mechanisms have been extensively investigated. In contrast, pattern formation from evaporating multicomponent systems, despite its wide existence in nature and numerous engineering applications, has been rarely explored. In this work, we examine a DNA-colloid binary suspension as a model system to understand the evaporation-induced interfacial hydrodynamics and self-assembled morphology in multicomponent systems involving complex competing intermolecular and interfacial interactions. Direct microscopic observations show that the composition of the binary system plays a critical role in the multiple-ring formation upon evaporation: (1) suspensions with high DNA concentrations and low colloidal concentrations favor the formation of the multiple-ring pattern; (2) the size of colloidal particles added into DNA aqueous droplets can significantly disrupt smooth multiple rings to form rippled rings and curtain-like periodic patterns with a curious spoke-like structure as the size of colloidal particles increases; and (3) the enhancement of DNA-colloid interaction by oppositely charged colloidal particles results in considerably high irregularity of DNA stain ring spacing. We examine the disruption of the multiring morphology under varied conditions and attribute it to local hydrodynamics governed by colloid aggregation and sedimentation. Our results demonstrate the feasibility of fabricating periodic self-assembled hybrid structures via one-step evaporation of droplets consisting of multiple components.     

Morphological Changes Induced by Addition of Polystyrene to Dextran-Polystyrene Block Copolymer Solutions

Summary: In this work we discuss the self-assembling behaviour in solution of a block copolymer, dextran-block-polystyrene, in the presence of homopolystyrene (PS) which allows to decrease the hydrophilic fraction f of the mixture. Dynamic and static light scattering experiments have been carried out in water-miscible solvents (DMSO and THF) to probe the formation of supramolecular structures. Results have been compared to those obtained with a block copolymer solution having the same hydrophilic fraction f. Interestingly, the morphology of the self-assembled structures was the same for a given value of f.