Self-assembly Propensity Research Articles

Refining the Design of Diblock Elastin-Like Polypeptides for Self-Assembly into Nanoparticles.

Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[M1V3-i]-[I-j]. First, ELP-[M1V3-i]-[I-j] (i = 20, 40, 60; j = 20, 90) that showed a similar self-assembly propensity (unimer-to-aggregate transition) as their related monoblocks ELP-[M1V3-i] and ELP-[I-j]. By selectively oxidizing methionines of ELP-[M1V3-i] within the different diblocks structures, we have been able to access a thermal phase transition with three distinct regimes (unimers, micelles, aggregates) characteristic of well-defined ELP diblocks.

Nanostructure, Self-Assembly, Mechanical Properties, and Antioxidant Activity of a Lupin-Derived Peptide Hydrogel.

Naturally occurring food peptides are frequently used in the life sciences due to their beneficial effects through their impact on specific biochemical pathways. Furthermore, they are often leveraged for applications in areas as diverse as bioengineering, medicine, agriculture, and even fashion. However, progress toward understanding their self-assembling properties as functional materials are often hindered by their long aromatic and charged residue-enriched sequences encrypted in the parent protein sequence. In this study, we elucidate the nanostructure and the hierarchical self-assembly propensity of a lupin-derived peptide which belongs to the α-conglutin (11S globulin, legumin-like protein), with a straightforward N-terminal biotinylated oligoglycine tag-based methodology for controlling the nanostructures, biomechanics, and biological features. Extensive characterization was performed via Circular Dichroism (CD) spectroscopy, Fourier Transform Infrared spectroscopy (FT-IR), rheological measurements, and Atomic Force Microscopy (AFM) analyses. By using the biotin tag, we obtained a thixotropic lupin-derived peptide hydrogel (named BT13) with tunable mechanical properties (from 2 to 11 kPa), without impairing its spontaneous formation of β-sheet secondary structures. Lastly, we demonstrated that this hydrogel has antioxidant activity. Altogether, our findings address multiple challenges associated with the development of naturally occurring food peptide-based hydrogels, offering a new tool to both fine tune the mechanical properties and tailor the antioxidant activities, providing new research directions across food chemistry, biochemistry, and bioengineering.

Unexpected Trends in Copper Removal from Aβ Peptide: When Less Ligand Is Better and Zn Helps.

Cu, Zn, and amyloid-β (Aβ) peptides play an important role in the etiology of Alzheimer's disease (AD). Their interaction indeed modifies the self-assembly propensity of the peptide that is at the origin of the deposition of insoluble peptide aggregates in the amyloid plaque, a hallmark found in AD brains. Another even more important fallout of the Cu binding to Aβ peptide is the formation of reactive oxygen species (ROS) that contributes to the overall oxidative stress detected in the disease and is due to the redox ability of the Cu ions. Many therapeutic approaches are currently developed to aid fighting against AD, one of them targeting the redox-active Cu ions. Along this research line, we report in the present article the use of a phenanthroline-based peptide-like ligand (L), which is able to withdraw Cu from Aβ and redox-silence it in a very stable 4N Cu(II) binding site even in the presence of Zn(II). In addition and in contrast to what is usually observed, the presence of excess of L lessens the searched effect of ROS production prevention, but it is counterbalanced by the co-presence of Zn(II). To explain such unprecedented trends, we proposed a mechanism that involves the redox reaction between Cu(II)L and Cu(I)L2. We thus illustrated (i) how speciation and redox chemistry can weaken the effect of a ligand that would have appeared perfectly suitable if only tested in a 1:1 ratio and on CuAβ and (ii) how Zn overcomes the undesired lessening of ROS arrest due to excess of ligand. In brief, we have shown how working in biologically relevant conditions is important for the understanding of all of the reactions at play and this must be taken into consideration for the further rational design of ligands aiming to become drug candidates.

Not Going with the Flow: How Cells Adapt Internal Physics

Defining the principles underlying the organization of biomolecules within cells is a key challenge of current cell biology research. Persson etal. now identify a powerful layer of regulation that allows cells to decouple diffusion from temperature by modulating their intracellular viscosity. This so-called viscoadaptation is mediated through trehalose and glycogen activities, which alter diffusion dynamics and self-assembly propensity inside the cell globally.

Double-Headed Cationic Lipopeptides: An Emerging Class of Antimicrobials.

Antimicrobial peptides (AMPs) constitute a promising tool in the development of novel therapeutic agents useful in a wide range of bacterial and fungal infections. Among the modifications improving pharmacokinetic and pharmacodynamic characteristics of natural AMPs, an important role is played by lipidation. This study focuses on the newly designed and synthesized lipopeptides containing multiple Lys residues or their shorter homologues with palmitic acid (C16) attached to the side chain of a residue located in the center of the peptide sequence. The approach resulted in the development of lipopeptides representing a model of surfactants with two polar headgroups. The aim of this study is to explain how variations in the length of the peptide chain or the hydrocarbon side chain of an amino acid residue modified with C16, affect biological functions of lipopeptides, their self-assembling propensity, and their mode of action.

Metal- and UV- Catalyzed Oxidation Results in Trapped Amyloid-β Intermediates Revealing that Self-Assembly Is Required for Aβ-Induced Cytotoxicity.

SummaryDityrosine (DiY), via the cross-linking of tyrosine residues, is a marker of protein oxidation, which increases with aging. Amyloid-β (Aβ) forms DiY in vitro and DiY-cross-linked Aβ is found in the brains of patients with Alzheimer disease. Metal- or UV- catalyzed oxidation of Aβ42 results in an increase in DiY cross-links. Using DiY as a marker of oxidation, we compare the self-assembly propensity and DiY cross-link formation for a non-assembly competent variant of Aβ42 (vAβ) with wild-type Aβ42. Oxidation results in the formation of trapped wild-type Aβ assemblies with increased DiY cross-links that are unable to elongate further. Assembly-incompetent vAβ and trapped Aβ assemblies are non-toxic to neuroblastoma cells at all stages of self-assembly, in contrast to oligomeric, non-cross-linked Aβ. These findings point to a mechanism of toxicity that necessitates dynamic self-assembly whereby trapped Aβ assemblies and assembly-incompetent variant Aβ are unable to result in cell death.

Spatiotemporal Self-Assembly of Peptides Dictates Cancer-Selective Toxicity

The intracellular or pericellular self-assembly of amphiphilic peptides is emerging as a potent cancer therapeutic strategy. Achieving the self-assembly of amphiphilic peptides inside a cell or cellular organelle is challenging due to the complex cellular environment, which consists of many amphiphilic biomolecules that may alter the self-assembling propensity of the synthetic peptides. Herein, we show that the hydrophobic-hydrophilic balance of the amphiphilic peptides determines the self-assembling propensity, thereby controlling the fate of the cell. A series of peptides were designed to target and self-assemble inside the mitochondria of cancer cells. The hydrophobicity of the peptides was tuned by varying their N-terminus capping. The analysis showed that the largest hydrophobic peptide was self-assembled before reaching the mitochondria and showed no selectivity toward cancer cells, whereas hydrophilic peptides could not self-assemble inside the mitochondria. Optimum balance between hydrophobicity and hydrophilicity is a critical factor for achieving self-assembly inside the mitochondria, thereby providing greater selectivity against cancer cells.

Impact of Mutations on the Conformational Transition from α-Helix to β-Sheet Structures in Arctic-Type Aβ40: Insights from Molecular Dynamics Simulations

The amyloid-β (Aβ) protein aggregation into toxic oligomers and fibrils has been recognized as a key player in the pathogenesis of Alzheimer’s disease. Recent experiments reported that a double alanine mutation (L17A/F19A) in the central hydrophobic core (CHC) region of [G22]Aβ40 (familial Arctic mutation) diminished the self-assembly propensity of [G22]Aβ40. However, the molecular mechanism behind the decreased aggregation tendency of [A17/A19/G22]Aβ40 is not well understood. Herein, we carried out molecular dynamics simulations to elucidate the structure and dynamics of [G22]Aβ40 and [A17/A19/G22]Aβ40. The results for the secondary structure analysis reveal a significantly increased amount of the helical content in the CHC and C-terminal region of [A17/A19/G22]Aβ40 as compared to [G22]Aβ40. The bending free-energy analysis of D23–K28 salt bridge suggests that the double alanine mutation in the CHC region of [G22]Aβ40 has the potential to reduce the fibril formation rate by 0.57 times of [G22]Aβ40. Unlike [G22]Aβ40, [A17/A19/G22]Aβ40 largely sampled helical conformation, as determined by the minimum energy conformations extracted from the free-energy landscape. The present study provided atomic level details into the experimentally observed diminished aggregation tendency of [A17/A19/G22]Aβ40 as compared to [G22]Aβ40.

Self-assembly of Alanine-Isoleucine and Isoleucine-Isoleucine Dipeptides through Atomistic Simulations and Experiments.

A detailed investigation of the structural and conformational properties of alanine-isoleucine (Ala-Ile) and isoleucine-isoleucine (Ile-Ile) dipeptides is presented in water and in methanol solvents. We propose a consistent combination of complementary simulation and experimental methods, covering a broad range of length and time scales, from the very short (i.e., atomic level), via all-atom molecular dynamics (MD) simulations, up to the macroscopic one, via scanning electron microscopy (SEM) experiments. The examined samples from both simulations and experiment cover a board range of concentrations since these are usually in different concentration windows (i.e., high values in simulations vs low values in experiments). In the present study, there is an overlapping concentration regime and a qualitative agreement between simulation and experimental results is observed. The effect of temperature on the formed structures is found to be small, from both simulation and experiments, when temperature varies from 278 to 300 K. Furthermore, the differences of Ala-Ile and Ile-Ile dipeptides from dialanine (Ala-Ala) and diphenylalanine (Phe-Phe) dipeptides in similar conditions are highlighted. Based on various measures, the strength of the self-assembly propensity of the four dipeptides in aqueous solutions attains the following order: Phe-Phe > Ala-Ile > Ala-Ala > Ile-Ile.

Structures of the intrinsically disordered Aβ, tau and α-synuclein proteins in aqueous solution from computer simulations

Intrinsically disordered proteins (IDPs) play many biological roles in the human proteome ranging from vesicular transport, signal transduction to neurodegenerative diseases. The Aβ and tau proteins, and the α-synuclein protein, key players in Alzheimer’s and Parkinson’s diseases, respectively are fully disordered at the monomer level. The structural heterogeneity of the monomeric and oligomeric states and the high self-assembly propensity of these three IDPs have precluded experimental structural determination. Simulations have been used to determine the atomic structures of these IDPs. In this article, we review recent computer models to capture the equilibrium ensemble of Aβ, tau and α-synuclein proteins at different association steps in aqueous solution and present new results of the PEP-FOLD framework on α-synuclein monomer.

Self-Assembly Propensity Dictates Lifetimes in Transient Naphthalimide-Dipeptide Nanofibers.

Transient self-assembly of dipeptide nanofibers with lifetimes that are predictably variable through dipeptide sequence design are presented. This was achieved using 1,8-naphthalimide (NI) amino acid methyl-esters (Phe, Tyr, Leu) that are biocatalytically coupled to amino acid-amides (Phe, Tyr, Leu, Val, Ala, Ser) to form self-assembling NI-dipeptides. However, competing hydrolysis of the dipeptides results in disassembly. It was demonstrated that the kinetic parameters like lifetimes of these nanofibers can be predictably regulated by the thermodynamic parameter, namely the self-assembly propensity of the constituent dipeptide sequence. These lifetimes could vary from minutes, to hours, to permanent gels that do not degrade. Moreover, the in-built NI fluorophore was utilized to image the transient nanostructures in solution with stimulated emission depletion (STED) based super-resolution fluorescence microscopy.

Coiled-coil inspired functional inclusion bodies

BackgroundRecombinant protein expression in bacteria often leads to the formation of intracellular insoluble protein deposits, a major bottleneck for the production of soluble and active products. However, in recent years, these bacterial protein aggregates, commonly known as inclusion bodies (IBs), have been shown to be a source of stable and active protein for biotechnological and biomedical applications. The formation of these functional IBs is usually facilitated by the fusion of aggregation-prone peptides or proteins to the protein of interest, leading to the formation of amyloid-like nanostructures, where the functional protein is embedded.ResultsIn order to offer an alternative to the classical amyloid-like IBs, here we develop functional IBs exploiting the coiled-coil fold. An in silico analysis of coiled-coil and aggregation propensities, net charge, and hydropathicity of different potential tags identified the natural homo-dimeric and anti-parallel coiled-coil ZapB bacterial protein as an optimal candidate to form assemblies in which the native state of the fused protein is preserved. The protein itself forms supramolecular fibrillar networks exhibiting only α-helix secondary structure. This non-amyloid self-assembly propensity allows generating innocuous IBs in which the recombinant protein of interest remains folded and functional, as demonstrated using two different fluorescent proteins.ConclusionsHere, we present a proof of concept for the use of a natural coiled-coil domain as a versatile tool for the production of functional IBs in bacteria. This α-helix-based strategy excludes any potential toxicity drawback that might arise from the amyloid nature of β-sheet-based IBs and renders highly active and homogeneous submicrometric particles.

Evaluating Self-Assembly Propensity of Tetra-Peptide using MD and Machine Learning

Evaluation of the self-assembly propensity such as aggregation and amyloid formation of a peptide is important to elucidate protein property. It is also useful in protein engineering for avoiding the aggregation of designed proteins. And, self-assembly structures with high stability are used for nanomaterials. As measuring aggregation propensity of many peptides using experimental methods are difficult, molecular dynamics (MD) simulation has been used for predicting self-assembling peptides. It has been reported that di-peptides (202=400 types) and tri-peptides (203=8000 types) aggregation propensity can be evaluated using MD simulation. However, such methods are difficult to be applied to tetra-peptides due to a large sequence space (204=160,000 types). In this study, we combined the MD simulation and machine learning to accelerate the evaluation of aggregation propensity. We conducted full atomistic MD simulation for 319 peptides and used them for training a machine-learning model to search all possible tetra-peptides. The two scores were used as the measures for self-assembly propensity: AP score related to the accessible surface area of peptides and Beta score related to the possibility of β-sheets structure. We validated that machine learning can accurately predict the result of MD simulation with Pearson's correlation coefficients of 0.88 (AP score) and 0.86 (Beta score). Top 100 predictions suggest that the histidine at C-terminus promotes the formation of a β-sheets structure. We experimentally confirmed that a selected peptide with N-terminal proline predicted to have high AP score and Beta score was aggregated and then changed to fibrous crystals. The crystal structure determined using X-ray diffraction data showed β-sheets structure. These results demonstrate that our framework combining machine learning and MD simulation is a promising strategy for evaluating a property of peptides and proteins.

Osmolyte-Induced Macromolecular Aggregation Is Length-Scale Dependent.

Cells survive in extreme environmental conditions by accumulating small organic molecules called osmolytes. While we have reached a consensus on the role of osmolytes toward macromolecular conformational stability at a single-macromolecule level, there is a lack of clarity on how osmolytes might influence macromolecular aggregation, an important feature to maintain cellular homeostasis. In this regard, here, we explore how a popular osmolyte trimethyl amine N-oxide (TMAO) individually dictates the self-assembling propensity of hydrophobic and charged macromolecules. Our computer simulation-based results reveal that the TMAO-induced self-aggregation of hydrophobic macromolecules, relative to that in neat water, is strongly dependent on the macromolecular length scale. Specifically, a free energy-based analysis indicates that the self-aggregation propensity of hydrophobic macromolecules in aqueous TMAO relative to neat water follows a nonmonotonic trend. When compared with neat water, TMAO promotes hydrophobic self-assembly at a shorter length scale while discourages hydrophobic self-assembly at a larger length scale. The overall nonmonotonic trend is found to be entropy driven. A molecular-level analysis suggests that length-scale-dependent preferential exclusion/binding of osmolytes (relative to water) from/to macromolecules in the process of aggregation holds the key to the nonmonotonic behavior. The overall result is found to be robust, even in the presence of the charge distributions on the macromolecules.

Catalyst: Can Systems Chemistry Unravel the Mysteries of the Chemical Origins of Life?

Daniela Kroiss obtained her MSc degree in biotechnology at BOKU and is currently a PhD student in the biochemistry program at CUNY, where she works on a joint project between the Nanoscience Initiative and the University of Strathclyde. Gonen Ashkenasy is head of the Laboratory for Systems Chemistry at the Ben-Gurion University Chemistry Department. His research focuses on the design and analysis of synthetic replication networks. Adam Braunschweig earned his PhD in organic chemistry at the University of California and is an associate professor in the CUNY Nanoscience Initiative. His research focuses on the interface of organic chemistry with biology and material science. Tell Tuttle obtained his PhD in theoretical chemistry at Goteborg University and is a professor of theoretical chemistry at the University of Strathclyde. His research focuses on the directed discovery of peptide-based materials. Rein Ulijn gained his PhD in physical chemistry at the University of Strathclyde and is founding director of the Nanoscience Initiative at the CUNY Advanced Science Research Center. His research focuses on nanotechnology inspired by living systems.

Molecular Recognition of Structures Is Key in the Polymerization of Patterned Barnacle Adhesive Sequences.

The permanent adhesive produced by adult barnacles is held together by tightly folded proteins that form amyloid-like materials distinct among marine foulants. In this work, we link stretches of alternating charged and noncharged linear sequences from a family of adhesive proteins to their role in forming fibrillar nanomaterials. Using recombinant proteins and short barnacle cement derived peptides (BCPs), we find a central sequence with charged motifs of the pattern [Gly/Ser/Val/Thr/Ala-X], where X are charged amino acids, to exert specific control over timing, structure, and morphology of fibril formation. While most BCPs remain dormant, the core segment demonstrates rapid polymerization as well as an ability to template other peptides with no propensity for self-assembly. Patterned charge domains assemble dormant peptides through a specific antiparallel β-sheet structure as measured by FTIR. While charged domains favor an antiparallel structure, BCPs without charged domains switch fibril assembly to favor simpler parallel β-sheet aggregates. In addition to activation, charged domains direct nanofibers to grow into discrete microns long fibrils similar to the natural adhesive, while segments without such domains only form short branched aggregates. The assembly of adhesive sequences through recognition of structured templates outlines a strategy used by barnacles to control physical mechanisms of underwater adhesive delivery, activation, and curing based on molecular recognition between proteins.

Synthesis and purification of self-assembling peptide-oligonucleotide conjugates by solid-phase peptide fragment condensation.

Peptide-oligonucleotide conjugates (POCs) are interesting molecules as they covalently combine 2 of the most important biomacromolecules. Sometimes, the synthesis of POCs involves unexpected difficulties; however, POCs with self-assembling propensity are even harder to synthesize and purify. Here, we show that solid-phase peptide fragment condensation combined with thiol-maleimide or copper-catalyzed azide-alkyne cycloaddition click chemistries is useful for the syntheses of self-assembling POCs. We describe guidelines for the selection of reactive functional groups and their placement during the conjugation reaction and consider the cost-effectiveness of the reaction. Purification is another important challenge during the preparation of POCs. Our results show that polyacrylamide gel electrophoresis under denaturing conditions is most suitable to recover a high yield of self-assembling POCs. This report provides the first comprehensive study of the preparation of self-assembling POCs, which will lay a foundation for the development of elegant and sophisticated molecular assemblies.

Structural Characterization of Self-Assembled Tetra-Tryptophan Based Nanostructures: Variations on a Common Theme.

Over the years, a large number of multidisciplinary investigations has unveiled that the self-assembly of short peptides and even of individual amino acids can generate a variety of different biomaterials. In this framework, we have recently reported that polyethylene glycol (PEG) conjugates of short homopeptides, containing aromatic amino acids such as phenylalanine (Phe, F) and naphthylalanine (Nal), are able to form elongated fibrillary aggregates having interesting chemical and physical properties. We here extend these analyses characterizing the self-assembling propensity of PEG6 -W4, a PEG adduct of the tetra-tryptophan (W4) sequence. A comprehensive structural characterization of PEG6 -W4 was obtained, both in solution and at the solid state, through the combination of spectroscopic, microscopic, X-ray scattering and computational techniques. Collectively, these studies demonstrate that this peptide is able to self-assemble in fibrillary networks characterized by a cross β-structure spine. The present findings clearly demonstrate that aromatic residues display a general propensity to induce self-aggregation phenomenon, despite the significant differences in the physicochemical properties of their side chains.

Ac-LVFFARK-NH2 conjugation to β-cyclodextrin exhibits significantly enhanced performance on inhibiting amyloid β-protein fibrillogenesis and cytotoxicity

Inhibition of amyloid β-protein (Aβ) aggregation is of significance for the potential treatment of Alzheimer's disease. We have herein conjugated heptapeptide Ac-LVFFARK-NH2 (LK7) to β-cyclodextrin (βCyD) and studied the inhibitory effect of the LK7-βCyD conjugate on Aβ aggregation. The conjugation significantly improved the peptide solubility and suppressed the self-assembly propensity. This led to 30% increase of the binding affinity of LK7 for Aβ in the conjugate due to increased hydrophobic interactions. Thus, LK7-βCyD suppressed the conformational transition of Aβ and showed stronger inhibitory effect on Aβ fibrillation than LK7. Thus, LK7-βCyD exhibited protective effect on Aβ40-induced cytotoxicity, and the cells completely survived at 10 molar excess of LK7-βCyD (from 67% to 100%). By contrast, LK7 showed only a moderate inhibition on Aβ fibrillation, and could not inhibit the amyloid cytotoxicity. The research proved that conjugation of hydrophobic peptide to βCyD was promising to increase its inhibition potency against Aβ aggregation.

The dominant role of side chains in supramolecular double helical organisation in synthetic tripeptides

Peptide self-assembled nanostructures have attracted attention recently owing to their promising applications in diversified avenues. To validate the importance of sidechains in supramolecular architectural stabilization, herein this report describes the self-assembly propensities involving weak interactions in a series of model tripeptides Boc-Xaa-Aib-Yaa-OMe I–IV, (where Xaa = 4-F-Phe/NMeSer/Ile & Yaa = Tyr in peptide I–III respectively and Xaa = 4-F-Phe & Yaa = Ile in peptide IV) differing in terminal side chains. The solid state structural analysis reveals that tripeptide (I) displays supramolecular preference for double helical architecture. However, when slight modification has been introduced in the N-terminal side chains disfavour the double helical organisation (Peptide II and III). Indeed the peptides display sheet like ensemble within the framework. Besides replacement of C-terminal Tyr by Ile in peptide I even do not promote the architecture, emphasizing the dominant role of balance of side chains in stabilizing double helical organisation. The CD measurements, concentration dependant studies, NMR titrations and ROESY spectra are well in agreement with the solid state conformational investigation. Moreover the morphological experiments utilizing FE-SEM, support the heterogeneity present in the peptides. Thus this work may not only hold future promise in understanding the structure and function of neurodegenerative diseases but also assist in rational design of protein modification in biologically active peptides.