Hexafluoroisopropanol induces self‐assembly of β‐amyloid peptides into highly ordered nanostructures

Deposition of insoluble fibrillar aggregates of β‐amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease. Apart from forming fibrils, these peptides also exist as soluble aggregates. Fibrillar and a variety of nonfibrillar aggregates of Aβ have also been obtained in vitro. Hexafluoroisopropanol (HFIP) has been widely used to dissolve Aβ and other amyloidogenic peptides. In this study, we show that the dissolution of Aβ40, 42, and 43 in HFIP followed by drying results in highly ordered aggregates. Although α‐helical conformation is observed, it is not stable for prolonged periods. Drying after prolonged incubation of Aβ40, 42, and 43 peptides in HFIP leads to structural transition from α‐helical to β‐conformation. The peptides form short fibrous aggregates that further assemble giving rise to highly ordered ring‐like structures. Aβ16–22, a highly amyloidogenic peptide stretch from Aβ, also formed very similar rings when dissolved in HFIP and dried. HFIP could not induce α‐helical conformation in Aβ16–22, and rings were obtained from freshly dissolved peptide. The rings formed by Aβ40, 42, 43, and Aβ16–22 are composed of the peptides in β‐conformation and cause enhancement in thioflavin T fluorescence, suggesting that the molecular architecture of these structures is amyloid‐like. Our results clearly indicate that dissolution of Aβ40, 42 and 43 and the amyloidogenic fragment Aβ16–22 in HFIP results in the formation of annular amyloid‐like structures. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Nanostructures from the self‐assembly of α‐helical peptide amphiphiles

Self‐assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO‐(IEEYTKK)_n‐NH₂ (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α‐helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α‐helical conformations under the tested conditions. The morphology of the self‐assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self‐assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self‐assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Organoids and the genetically encoded self‐assembly of embryonic stem cells

Understanding the mechanisms of early embryonic patterning and the timely allocation of specific cells to embryonic regions and fates as well as their development into tissues and organs, is a fundamental problem in Developmental Biology. The classical explanation for this process had been built around the notion of positional information. Accordingly the programmed appearance of sources of Morphogens at localized positions within a field of cells directs their differentiation. Recently, the development of organs and tissues from unpatterned and initially identical stem cells (adult and embryonic) has challenged the need for positional information and even the integrity of the embryo, for pattern formation. Here we review the emerging area of organoid biology from the perspective of Developmental Biology. We argue that the events underlying the development of these systems are not purely linked to “self‐organization,” as often suggested, but rather to a process of genetically encoded self‐assembly where genetic programs encode and control the emergence of biological structures.     

Homo‐ and heteropolymer self‐assembly of recombinant trichocytic keratins

In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues. However, during extraction from natural sources, such as hair and wool fibers, natural keratins are subject to extensive processing conditions that lead to formation of unwanted by‐products. Additionally, natural keratins suffer from limited sequence tunability. Recombinant keratin proteins can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Herein, we present the bacterial expression, purification, and solution characterization of human hair keratins K31 and K81. The obligate heterodimerization of the K31/K81 pair that results in formation of intermediate filaments is maintained in the recombinant proteins. Surprisingly, we have for the first time observed new zero‐ and one‐dimensional nanostructures from homooligomerization of K81 and K31, respectively. Further analysis of the self‐assembly mechanism highlights the importance of disulfide crosslinking in keratin self‐assembly.     

Modeling the competition between aggregation and self‐assembly during virus‐like particle processing

Understanding and controlling aggregation is an essential aspect in the development of pharmaceutical proteins to improve product yield, potency and quality consistency. Even a minute quantity of aggregates may be reactogenic and can render the final product unusable. Self‐assembly processing of virus‐like particles (VLPs) is an efficient method to quicken the delivery of safe and efficacious vaccines to the market at low cost. VLP production, as with the manufacture of many biotherapeutics, is susceptible to aggregation, which may be minimized through the use of accurate and practical mathematical models. However, existing models for virus assembly are idealized, and do not predict the non‐native aggregation behavior of self‐assembling viral subunits in a tractable nor useful way. Here we present a mechanistic mathematical model describing VLP self‐assembly that accounts for partitioning of reactive subunits between the correct and aggregation pathways. Our results show that unproductive aggregation causes up to 38% product loss by competing favorably with the productive nucleation of self‐assembling subunits, therefore limiting the availability of nuclei for subsequent capsid growth. The protein subunit aggregation reaction exhibits an apparent second‐order concentration dependence, suggesting a dimerization‐controlled agglomeration pathway. Despite the plethora of possible assembly intermediates and aggregation pathways, protein aggregation behavior may be predicted by a relatively simple yet realistic model. More importantly, we have shown that our bioengineering model is amenable to different reactor formats, thus opening the way to rational scale‐up strategies for products that comprise biomolecular assemblies. Biotechnol. Bioeng. 2010;107: 550–560. © 2010 Wiley Periodicals, Inc.     

Self‐assembly of pH and calcium dual‐responsive peptide‐amphiphilic hydrogel

Peptide‐based hydrogels have gained much interest for biomedical applications as a result of their biocompatibility. Herein, we reported a synthetic pH‐sensitive and calcium‐responsive peptide‐amphiphilic hydrogel. The sequences of the peptide amphiphiles were derived from the repeat‐in‐toxin (RTX) motif. At a certain peptide‐amphiphile concentration, self‐assembly was accompanied by the formation of a rigid, viscoelastic hydrogel at low pH or the presence of calcium ions. Circular dichroism spectra showed that the peptide amphiphiles adopted beta‐sheet structure. Meanwhile, as revealed by transmission electron microscopy, the peptide‐amphiphile self‐assembly was accompanied by the formation of long interconnected nanofibrillar superstructure. Material properties of the resulting peptide‐amphiphile hydrogel were characterized using oscillatory sheer rheology, and the storage modulus (G′) was found to be one order of magnitude higher than the loss modulus (G″), indicating a moderately rigid viscoelastic material. Furthermore, with systematical residue substitution, it was found that the aspartic acid within the repeat‐in‐toxin sequence of peptide amphiphiles was responsible for the pH and calcium selectivity. The environmental responsiveness, secondary structure, morphology, and mechanical nature of the peptide‐amphiphile hydrogel make it a possible material candidate for biomedical and engineering application. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Distinct solid and solution state self‐assembly pathways of RADA16‐I designer peptide

Solid state NMR measurements on selectively ¹³C‐labeled RADA16‐I peptide (COCH₃–RADARADARADARADA–NH₂) were used to obtain new molecular level information on the conversion of α‐helices to β‐sheets through self‐assembly in the solid state with increasing temperature. Isotopic labeling at the A4 C_β site enabled rapid detection of ¹³C NMR signals. Heating to 344–363 K with simultaneous NMR detection allowed production of samples with systematic variation of α‐helix and β‐strand content. These samples were then probed at room temperature for intermolecular ¹³C–¹³C nuclear dipolar couplings with the PITHIRDS‐CT NMR experiment. The structural transition was also characterized by Fourier transform infrared spectroscopy and wide angle X‐ray diffraction. Independence of PITHIRDS‐CT decay shapes on overall α‐helical and β‐strand content infers that β‐strands are not observed without association with β‐sheets, indicating that β‐sheets are formed at elevated temperatures on a timescale that is fast relative to the NMR experiment. PITHIRDS‐CT NMR data were compared with results of similar measurements on RADA16‐I nanofibers produced by self‐assembly in aqueous salt solution. We report that β‐sheets formed through self‐assembly in the solid state have a structure that differs from those formed through self‐assembly in the solution state. Specifically, solid state RADA16‐I self‐assembly produces in‐register parallel β‐sheets, whereas nanofibers are composed of stacked parallel β‐sheets with registry shifts between adjacent β‐strands in each β‐sheet. These results provide evidence for environment‐dependent self‐assembly mechanisms for RADA16‐I β‐sheets as well as new constraints on solid state self‐assembled structures, which must be avoided to maximize solution solubility and nanofiber yields. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Coevolutionary constraints in the sequence‐space of macromolecular complexes reflect their self‐assembly pathways

Is the order in which biomolecular subunits self‐assemble into functional macromolecular complexes imprinted in their sequence‐space? Here, we demonstrate that the temporal order of macromolecular complex self‐assembly can be efficiently captured using the landscape of residue‐level coevolutionary constraints. This predictive power of coevolutionary constraints is irrespective of the structural, functional, and phylogenetic classification of the complex and of the stoichiometry and quaternary arrangement of the constituent monomers. Combining this result with a number of structural attributes estimated from the crystal structure data, we find indications that stronger coevolutionary constraints at interfaces formed early in the assembly hierarchy probably promotes coordinated fixation of mutations that leads to high‐affinity binding with higher surface area, increased surface complementarity and elevated number of molecular contacts, compared to those that form late in the assembly. Proteins 2017; 85:1183–1189. © 2017 Wiley Periodicals, Inc.     

Bidirectional solid phase synthesis of a model oligoglycine bolaamphiphile and purification by rapid self‐assembly

We utilised a simple bidirectional (N→C and C→N) solid phase synthesis strategy entailing conventional solid phase peptide synthesis and fragment condensation with a water‐soluble carbodiimide to synthesise a model anionic glycylglycine bolaamphiphile containing a suberic acid linker moiety, namely N,N′‐suberoyldiglycylglycine. The synthetic suberoyldiglycylglycine was purified using its inherent ability to rapidly self‐assemble in an aqueous acidic solution (0.1% trifluoroacetic acid). Monitoring of the rapid assembly process corroborated our visual observation and confirmed packing‐directed self‐assembly rather than non‐specific aggregation or precipitation. The progress of suberoyldiglycylglycine self‐assembly was observed to be via the formation of oligomers in the solution, which then self‐assembled to form layered β‐sheet type macrostructures. Within 24 h, nanotubes grew from these macrostructures and eventually combined to formed microtubes, which we isolated after 5–7 days. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Solution structure and interface‐driven self‐assembly of NC2, a new member of the Class II hydrophobin proteins

Hydrophobins are fungal proteins that self‐assemble spontaneously to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin‐based applications. Proteins 2014; 82:990–1003. © 2013 Wiley Periodicals, Inc.     

A novel self‐assembling peptide with UV‐responsive properties

A novel heptapeptide comprising Ile‐Gln‐Ser‐Pro‐His‐Phe‐Phe (IQSPHFF) identified and found to undergo self‐assembly into microparticles in solution. To understand the effects of ultraviolet (UV) irradiation on the self‐assembly process, IQSPHFF solutions were exposed to the UV light of 365 nm at room temperature. This exposure was found to have a profound effect on the morphology of the self‐assembled aggregates, converting the microparticles to nanorod shapes. Circular dichroism and FTIR studies indicated distinct structural differences in the arrangements of the peptide moieties before and after UV irradiation. However, Mass spectrum analysis and high performance liquid chromatography of the peptide molecules before and after UV irradiation demonstrated that the chemical structure of IQSPHFF was not changed. UV–visible spectroscopy and fluorescence spectroscopy studies showed that the absorption peak both increased after UV irradiation. Overall, our data show that the heptapeptide with UV‐responsive properties. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 272–278, 2014.     

Helicity of perfluoroalkyl chains controlled by the self‐assembly of the Ala‐Ala dipeptides

Four Ala‐Ala dipeptides with a perfluoroalkyl chain at the N‐terminal were synthesized. They were able to self‐assemble into helical nanofibers and/or twisted nanobelts in a mixture of DMSO/H₂O. The handedness of nanofibers and nanobelts was controlled by the chirality of the alanine at the N‐terminal. The stacking handedness of the phenylene groups and the helicity of the perfluoroalkyl chain were studied using circular dichroism spectroscopy and vibrational circular dichroism, respectively. The chirality of the alanine at N‐terminal controlled the stacking handedness of the neighboring phenylene groups. Moreover, due to the low potential barrier between M‐ and P‐helices of the perfluorocarbon chain, the handedness of the organic self‐assemblies eventually controlled the helicity of the perfluorocarbon chain. X‐ray diffraction indicated that a lamellar structure was formed by the dimers of the dipeptides.     

The predominant roles of the sequence periodicity in the self‐assembly of collagen‐mimetic mini‐fibrils

Collagen fibrils represent a unique case of protein folding and self‐association. We have recently successfully developed triple‐helical peptides that can further self‐assemble into collagen‐mimetic mini‐fibrils. The 35 nm axially repeating structure of the mini‐fibrils, which is designated the d‐period, is highly reminiscent of the well‐known 67 nm D‐period of native collagens when examined using TEM and atomic force spectroscopy. We postulate that it is the pseudo‐identical repeating sequence units in the primary structure of the designed peptides that give rise to the d‐period of the quaternary structure of the mini‐fibrils. In this work, we characterize the self‐assembly of two additional designed peptides: peptide Col877 and peptide Col108rr. The triple‐helix domain of Col877 consists of three pseudo‐identical amino acid sequence units arranged in tandem, whereas that of Col108rr consists of three sequence units identical in amino acid composition but different in sequence. Both peptides form stable collagen triple helices, but only triple helices Col877 self‐associate laterally under fibril forming conditions to form mini‐fibrils having the predicted d‐period. The Co108rr triple helices, however, only form nonspecific aggregates having no identifiable structural features. These results further accentuate the critical involvement of the repeating sequence units in the self‐assembly of collagen mini‐fibrils; the actual amino acid sequence of each unit has only secondary effects. Collagen is essential for tissue development and function. This novel approach to creating collagen‐mimetic fibrils can potentially impact fundamental research and have a wide range of biomedical and industrial applications.     

A proposed model for dragline spider silk self‐assembly: Insights from the effect of the repetitive domain size on fiber properties

Dragline spider silk has been intensively studied for its superior qualities as a biomaterial. In previous studies, we made use of the baculovirus mediated expression system for the production of a recombinant Araneus diadematus spider silk dragline ADF4 protein and its self‐assembly into intricate fibers in host insect cells. In this study, our aim was to explore the function of the major repetitive domain of the dragline spider silk. Thus, we generated an array of synthetic proteins, each containing a different number of identical repeats up to the largest recombinantly expressed spider silk to date. Study of the self‐assembly properties of these proteins showed that depending on the increasing number of repeats they give rise to different assembly phenotypes, from a fully soluble protein to bona fide fibers with superior qualities. The different assembly forms, the corresponding chemical resistance properties obtained as well as ultrastructural studies, revealed novel insights concerning the structure and intermolecular interactions of the repetitive and nonrepetitive domains. Based on these observations and current knowledge in the field, we hereby present a comprehensive hypothetical model for the mechanism of dragline silk self‐assembly and fiber formation. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 458–468, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Bioproduction and characterization of a pH responsive self‐assembling peptide

Peptide P₁₁‐4 (QQRFEWEFEQQ) was designed to self‐assemble to form β‐sheets and nematic gels in the pH range 5–7 at concentrations ≥12.6 mM in water. This self‐assembly is reversibly controlled by adjusting the pH of the solvent. It can also self‐assemble into gels in biological media. This together with its biocompatibility and biodegradability make P₁₁‐4 an attractive building block for the fabrication of nanoscale materials with uses in, for example, tissue engineering. A limitation to large‐scale production of such peptides is the high cost of solid phase chemical synthesis. We describe expression of peptide P₁₁‐4 in the bacterium Escherichia coli from constructs carrying tandem repeats of the peptide coding sequence. The vector pET31b+ was used to express P₁₁‐4 repeats fused to the ketosteroid isomerase protein which accumulates in easily recoverable inclusion bodies. Importantly, the use of auto‐induction growth medium to enhance cell density and protein expression levels resulted in recovery of 2.5 g fusion protein/L culture in both shake flask and batch fermentation. Whole cell detergent lysis allowed recovery of inclusion bodies largely composed of the fusion protein. Cyanogen bromide cleavage followed by reverse phase HPLC allowed purification of the recombinant peptide with a C‐terminal homoserine lactone (rP₁₁‐4(hsl)). This recombinant peptide formed pH dependent hydrogels, displayed β‐structure measured by circular dichroism and fibril formation observed by transmission electron microscopy. Biotechnol. Bioeng. 2009;103: 241–251. © 2009 Wiley Periodicals, Inc.     

Supramolecular assembly of collagen triblock peptides

The relationship between primary sequence and collagen triple-helix formation is relatively well characterized, while higher levels of structural assembly from these sequences is poorly understood. To address this gap, a new collagen-like triblock peptide design was used to study the relationship between amino acid sequence and supramolecular assembly. Four collagen-like peptides with the sequence (Glu)(5)(Gly-Xaa-Hyp-Gly-Pro-Hyp)(6)(Glu)(5) and corresponding to Xaa = alanine, proline, serine, or valine, and an analogous peptide without the glutamic acid end blocks, were solubilized in water at high concentrations (20-150 mg/mL) and analyzed in optical polarizing microscopy and transmission electron microscopy. Some of the peptides self-assembled into supramolecular structures, the nature of which was determined by the core collagen-like sequence. The globular end blocks appeared necessary for these short triple-helix-forming peptides to spontaneously organize into supramolecular structures in solution and also provided enhanced thermal stability based on CD analysis. The results indicate a strong dependence of the peptide triblock assembly behavior on the identity of the guest residue Xaa; nematic order when Xaa was valine, no organization when Xaa was serine, and banded spherulites displaying a cholesteric-like twist when Xaa was proline or alanine. According to these results, the identity of the amino acid in position Xaa of the triplet Gly-Xaa-Yaa dramatically determined the type of supramolecular assembly formed by short triple helices based on collagen-triblock like sequences. Moreover, the structural organization observed for these collagen-triblock peptides was analogous to some assemblies observed for native collagen in vivo and in vitro. The amino acid sequence in the native collagen proteins may therefore be a direct determinant of the different supramolecular architectures found in connective tissues.     

To achieve self‐assembled collagen mimetic fibrils using designed peptides

It has proven challenging to obtain collagen‐mimetic fibrils by protein design. We recently reported the self‐assembly of a mini‐fibril showing a 35 nm, D‐period like, axially repeating structure using the designed triple helix Col108. Peptide Col108 was made by bacterial expression using a synthetic gene; its triple helix domain consists of three pseudo‐identical units of amino acid sequence arranged in tandem. It was postulated that the 35 nm d‐period of Col108 mini‐fibrils originates from the periodicity of the Col108 primary structure. A mutual staggering of one sequence unit of the associating Col108 triple helices can maximize the inter‐helical interactions and produce the observed 35 nm d‐period. Based on this unit‐staggered model, a triple helix consisting of only two sequence units is expected to have the potential to form the same d‐periodic mini‐fibrils. Indeed, when such a peptide, peptide 2U108, was made it was found to self‐assemble into mini‐fibrils having the same d‐period of 35 nm. In contrast, no d‐periodic mini‐fibrils were observed for peptide 1U108, which does not have long‐range repeating sequences in its primary structure. The findings of the periodic mini‐fibrils of Col108 and 2U108 suggest a way forward to create collagen‐mimetic fibrils for biomedical and industrial applications.