Directed intermixing in multicomponent self-assembling biomaterials

The noncovalent coassembly of multiple different peptides can be a useful route for producing multifunctional biomaterials. However, to date, such materials have almost exclusively been investigated as homogeneous self-assemblies, having functional components uniformly distributed throughout their supramolecular structures. Here we illustrate control over the intermixing of multiple different self-assembling peptides, in turn providing a simple but powerful means for modulating these materials' mechanical and biological properties. In β-sheet fibrillizing hydrogels, significant increases in stiffening could be achieved using heterobifunctional cross-linkers by sequestering peptides bearing different reactive groups into distinct populations of fibrils, thus favoring interfibril cross-linking. Further, by specifying the intermixing of RGD-bearing peptides in 2-D and 3-D self-assemblies, the growth of HUVECs and NIH 3T3 cells could be significantly modulated. This approach may be immediately applicable toward a wide variety of self-assembling systems that form stable supramolecular structures.     

Short self-assembling peptides as building blocks for modern nanodevices

Short, self-assembling peptides form a variety of stable nanostructures used for the rational design of functional devices. Peptides serve as organic templates for conjugating biorecognition elements, and assembling ordered nanoparticle arrays and hybrid supramolecular structures. We are witnessing the emergence of a new phase of bionanotechnology, particularly towards electronic, photonic and plasmonic applications. Recent advances include self-assembly of photoluminescent semiconducting nanowires and peptide-conjugated systems for sensing, catalysis and energy storage. Concurrently, methods and tools have been developed to control and manipulate the self-assembled nanostructures. Furthermore, there is growing knowledge on nanostructure properties such as piezoelectricity, dipolar electric field and stability. This review focuses on the emerging role of short, linear self-assembling peptides as simple and versatile building blocks for nanodevices.     

智能多肽的自组装机理及其生物学应用

智能多肽是指智能响应外界刺激并做出相应回应的多肽。由于其形成过程为自发的自组装,故智能多肽又可称为自组装多肽。智能多肽的氨基酸构成使其拥有良好的生物相容性及生物可降解性,作为构筑基元拼接成为功能性材料,在新型生物材料方面展示出了广阔的应用前景。概括了智能多肽的性质、自组装机理及应用,重点阐述了它在生物能源、生物医学工程和分离工程上的应用,以期在系统认识智能多肽的基础上,发掘其应用潜能,突破开发瓶颈。     

Heterochiral tetrapeptide self-assembly into hydrogel biomaterials for hydrolase mimicry

Self-assembling short peptides have attracted great interest as enzyme mimics, especially if the catalytic activity resides solely in the supramolecular structure so that it can be switched on/off as needed by controlling assembly/disassembly. Among the various enzyme classes, hydrolases find wide application in biomaterials, and their mimetics often contain His residues, in addition to either divalent cations or other amino acids to mimic the catalytic site. This work reports two self-assembling tetrapeptides based on the Ser-His motif for catalysis and the Phe-Phe motif to drive amyloid structure formation. Both peptides form thermoreversible hydrogels in phosphate buffer at neutral pH that display a mild esterase-like activity, as demonstrated on the hydrolysis of 4-nitrophenyl acetate as a model substrate, although presence of Ser did not enhance catalytic activity. The systems are characterised by circular dichroism, transmission electron microscopy, oscillatory rheology and Thioflavin T fluorescence as an amyloid stain, to provide further insights that may assist the future design of improved supramolecular catalysts.     

De novo design of α,β-didehydrophenylalanine containing peptides: from models to applications

The de novo design of peptides and proteins has emerged as an approach for investigating protein structure and function. The success relies heavily on the ability to design relatively short peptides that can adopt stable secondary structures. To this end, substitution with α,β-dehydroamino acids, especially α,β-didehydrophenylalanine (ΔPhe or ΔF) has blossomed in manifold directions, providing a rich diversity of well-defined structural motifs. Introduction of α,β-didehydrophenylalanine induces β-bends in small and 3(10)-helices in longer peptide sequences. Most favorable conformation of ΔF residues are (φ,ψ) ～(60°, 30°), (-60°, -30°), (-60°, 150°), and (60°, -150°). These features have been exploited in designing helix-turn-helix, helical bundle arrangements, and glycine zipper type super secondary structural motifs. The unusual capability of α,β-didehydrophenylalanine ring to form a variety of multicentered interactions (N-H…O, C-H…O, C-H…π, and N-H…π) suggests its possible exploitation for future de novo design of supramolecular structures. This work has now been extended to the de novo design of peptides with antibiotic, antifibrillization activity, etc. More recently, self-assembling properties of small dehydropeptides have been explored. This review focuses primarily on the structural and functional behavior of α,β-didehydrophenylalanine containing peptides.     

Modulation of hydrogel networks by metal ions

Self-assembling hydrogels are receiving great attention for both biomedical and technological applications. Self-assembly of protein/peptides as well as organic molecules is commonly induced in response to external triggers such as changes of temperature, concentration, or pH. An interesting strategy to modulate the morphology and mechanical properties of the gels implies the use of metal ions, where coordination bonds regulate the dynamic cross-linking in the construction of hydrogels, and coordination geometries, catalytic, and redox properties of metal ions play crucial roles. This review aims to discuss recent insights into the supramolecular assembly of hydrogels involving metal ions, with a focus on self-assembling peptides, as well as applications of metallogels in biomedical fields including tissue engineering, sensing, wound healing, and drug delivery.     

Creating Prebiotic Sanctuary: Self-Assembling Supramolecular Peptide Structures Bind and Stabilize RNA

Any attempt to uncover the origins of life must tackle the known ‘blind watchmaker problem’. That is to demonstrate the likelihood of the emergence of a prebiotic system simple enough to be formed spontaneously and yet complex enough to allow natural selection that will lead to Darwinistic evolution. Studies of short aromatic peptides revealed their ability to self-assemble into ordered and stable structures. The unique physical and chemical characteristics of these peptide assemblies point out to their possible role in the origins of life. We have explored mechanisms by which self-assembling short peptides and RNA fragments could interact together and go through a molecular co-evolution, using diphenylalanine supramolecular assemblies as a model system. The spontaneous formation of these self-assembling peptides under prebiotic conditions, through the salt-induced peptide formation (SIPF) pathway was demonstrated. These peptide assemblies possess the ability to bind and stabilize ribonucleotides in a sequence-depended manner, thus increase their relative fitness. The formation of these peptide assemblies is dependent on the homochirality of the peptide monomers: while homochiral peptides (L-Phe-L-Phe and D-Phe-D-Phe) self-assemble rapidly in aqueous environment, heterochiral diastereoisomers (L-Phe-D-Phe and D-Phe-L-Phe) do not tend to self-assemble. This characteristic consists with the homochirality of all living matter. Finally, based on these findings, we propose a model for the role of short self-assembling peptides in the prebiotic molecular evolution and the origin of life.     

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.     

Enzymatic modification of self-assembled peptide structures with tissue transglutaminase

A de novo peptide that self-assembles into fibrillar structures and serves as a substrate for the cross-linking enzyme tissue transglutaminase was developed (Ac-QQKFQFQFEQQ-Am). Congo red staining, circular dichroism, and FTIR spectroscopy showed that this 11-amino acid peptide produced predominantly beta-sheet structures. TEM with negative staining and quick-freeze deep etch (QFDE) TEM showed that the peptide structures were composed of a highly entangled fibrillar network. These beta-sheet fibrillar nanostructures were then covalently coupled to pendant amine-containing biomolecules via tissue transglutaminase. MALDI-TOF mass spectrometry and HPLC were utilized to monitor the extent of the transglutaminase modification of the peptide, showing that as many as five glutamines in the peptide were reactive via transglutaminase for covalent conjugation. This strategy, based on the post-assembly modification of a self-assembling peptide, has potential applications for tailoring supramolecular structures for drug delivery, tissue engineering, or other biomedical applications.     

The use of supramolecular structures as protein ligands

Congo red dye as well as other eagerly self-assembling organic molecules which form rod-like or ribbon-like supramolecular structures in water solutions, appears to represent a new class of protein ligands with possible wide-ranging medical applications. Such molecules associate with proteins as integral clusters and preferentially penetrate into areas of low molecular stability. Abnormal, partly unfolded proteins are the main binding target for such ligands, while well packed molecules are generally inaccessible. Of particular interest is the observation that local susceptibility for binding supramolecular ligands may be promoted in some proteins as a consequence of function-derived structural changes, and that such complexation may alter the activity profile of target proteins. Examples are presented in this paper.     

Supramolecular ligands: Monomer structure and protein ligation capability

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of his azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to his azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.     

Acceleration of protein aggregation by amphiphilic peptides: transformation of supramolecular structure of the aggregates

Protein self-assembly and aggregation represent a special tool in biomedicine and biotechnology to produce biological materials for a wide range of applications. The protein aggregates are very different morphologically, varying from soluble amorphous aggregates to highly ordered amyloid-like fibrils, the latter being associated with molecular structures able to perform specific functions in living systems. Fabrication of novel biomaterials resembling natural protein assemblies has awakened interest in identification of low-molecular-weight biogenic agents as regulators of transformation of aggregation-prone proteins into fibrillar structures. Short amphiphilic peptides can be considered for this role. Using dynamic light scattering, turbidimetry, fluorescence spectroscopy, and transmission electron microscopy (TEM), we have demonstrated that the Arg-Phe dipeptide dramatically accelerates the aggregation of a model protein, α-lactalbumin, to generate morphologically different structures. TEM revealed transformation of spherical particles observed in the control samples into branched chains of fibril-like nanostructures in the presence of the peptide, suggesting that amphiphilic peptides can induce changes in the physicochemical properties of a protein substrate (net charge, hydrophobicity, and tendency to β-structure formation) resulting in accumulation of peptide-protein complexes competent to self-assembly into supramolecular structures. A number of other short amphiphilic peptides have also been shown to accelerate the aggregation process, using alternative complementary protein substrates for identification of molecular recognition modules. Peptide-protein assemblies are suggested to play the role of building blocks for formation of supramolecular structures profoundly differing from those of the individual protein substrate in type, size, and shape.     

Monomer-monomer interactions propagate structural transitions necessary for pore formation by the cholesterol-dependent cytolysins

The assembly of the cholesterol-dependent cytolysin (CDC) oligomeric pore complex requires a complex choreography of secondary and tertiary structural changes in domain 3 (D3) of the CDC monomer structure. A point mutation was identified in the archetype CDC, perfringolysin O, that blocks detectable D3 structural changes and traps the membrane-bound monomers in an early and reversible stage of oligomer assembly. Using this and other mutants we show that specific D3 structural changes are propagated from one membrane-bound monomer to another. Propagation of these structural changes results in the exposure of a β-strand in D3 that allows it to pair and form edge-on interactions with a second β-strand of a free membrane-bound monomer. Pairing of these strands establishes the final geometry of the pore complex and is necessary to drive the formation of the β-barrel pore. These studies provide new insights into how structural information is propagated between membrane-bound monomers of a self-assembling system and the interactions that establish the geometry of the final pore complex.     

Novel peptide-based biomaterial scaffolds for tissue engineering.

Biomaterial scaffolds are components of cell-laden artificial tissues and transplantable biosensors. Some of the most promising new synthetic biomaterial scaffolds are composed of self-assembling peptides that can be modified to contain biologically active motifs. Peptide-based biomaterials can be fabricated to form two- and three-dimensional structures. Recent studies show that biomaterial promotion of multi-dimensional cell-cell interactions and cell density are crucial for proper cellular differentiation and for subsequent tissue formation. Other refinements in tissue engineering include the use of stem cells, cell pre-selection and growth factor pre-treatment of cells that are used for seeding scaffolds. These cell-culture technologies, combined with improved processes for defining the dimensions of peptide-based scaffolds, might lead to further improvements in tissue engineering. Novel peptide-based biomaterial scaffolds seeded with cells show promise for tissue repair and for other medical applications.     

Self-assembly of amphipathic β-sheet peptides: insights and applications

Amphipathic peptides composed of alternating polar and nonpolar residues have a strong tendency to self-assemble into one-dimensional, amyloid-like fibril structures. Fibrils derived from peptides of general (XZXZ)(n) sequence in which X is hydrophobic and Z is hydrophilic adopt a putative β-sheet bilayer. The bilayer configuration allows burial of the hydrophobic X side chain groups in the core of the fibril and leaves the polar Z side chains exposed to solvent. This architectural arrangement provides fibrils that maintain high solubility in water and has facilitated the recent exploitation of self-assembled amphipathic peptide fibrils as functional biomaterials. This article is a critical review of the development and application of self-assembling amphipathic peptides with a focus on the fundamental insight these types of peptides provide into peptide self-assembly phenomena.     

Enhancement of HIV-1 infectivity by simple, self-assembling modular peptides

Semen-derived enhancer of viral infection (SEVI), an amyloid fibril formed from a cationic peptide fragment of prostatic acidic phosphatase (PAP), dramatically enhances the infectivity of human immunodeficiency virus type 1 (HIV-1). Insoluble, sedimentable fibrils contribute to SEVI-mediated enhancement of virus infection. However, the SEVI-forming PAP(248–286) peptide is able to produce infection-enhancing structures much more quickly than it forms amyloid fibrils. This suggests that soluble supramolecular assemblies may enhance HIV-1 infection. To address this question, non-SEVI amyloid-like fibrils were derived from general amphipathic peptides of sequence Ac-K_n(XKXE)₂-NH₂. These cationic peptides efficiently self-assembled to form soluble, fibril-like structures that were, in some cases, able to enhance HIV-1 infection even more efficiently than SEVI. Experiments were also performed to determine whether agents that efficiently shield the charged surface of SEVI fibrils block SEVI-mediated infection-enhancement. To do this, we generated self-assembling anionic peptides of sequence Ac-E_n(XKXE)₂-NH₂. One of these peptides completely abrogated SEVI-mediated enhancement of HIV-1 infection, without altering HIV-1 infectivity in the absence of SEVI. Collectively, these data suggest that soluble SEVI assemblies may mediate infection-enhancement, and that anionic peptide supramolecular assemblies have the potential to act as anti-SEVI microbicides.     

Formation of framework nacre polypeptide supramolecular assemblies that nucleate polymorphs

The formation of aragonite in the mollusk shell nacre layer is linked to the assembly of framework protein complexes that interact with β-chitin polysaccharide. What is not yet understood is how framework nacre proteins control crystal growth. Recently, a 30 AA intrinsically disordered nacre protein sequence (n16N) derived from the n16 framework nacre protein was found to form aragonite, vaterite, or ACC deposits when adsorbed onto β-chitin. Our present study now establishes that n16N assembles to form amorphous nonmineralized supramolecular complexes that nucleate calcium carbonate polymorphs in vitro. These complexes contain unfolded or disordered (54% random coil, 46% β structures) n16N polypeptide chains that self-assemble in response to alkaline pH shift. The pH-dependent assembly process involves two stages, and it is likely that side chain salt-bridging interactions are a major driving force in n16N self-association. Intriguingly, Ca(II) ions are not required for n16N assembly but do shift the assembly process to higher pH values, and it is likely that Ca(II) plays some role in stabilizing the monomeric form of n16N. Using preassembled fibril-spheroid n16N assemblies on Si wafers or polystyrene supports, we were able to preferentially nucleate vaterite at higher incidence compared to control scenarios, and it is clear that the n16N assemblies are in contact with the nucleating crystals. We conclude that the framework nacre protein sequence n16N assembles to form supramolecular complexes whose surfaces act as nucleation sites for crystal growth. This may represent a general mineralization mechanism employed by framework nacre proteins in general.     

Investigating the effects of N-terminal acetylation on KFE8 self-assembly with 2D IR spectroscopy

Peptide self-assembly is an exciting and robust approach to create novel nanoscale materials for biomedical applications. However, the complex interplay between intra- and intermolecular interactions in peptide aggregation means that minor changes in peptide sequence can yield dramatic changes in supramolecular structure. Here, we use two-dimensional infrared spectroscopy to study a model amphiphilic peptide, KFE8, and its N-terminal acetylated counterpart, AcKFE8. Two-dimensional infrared spectra of isotope-labeled peptides reveal that AcKFE8 aggregates comprise two distinct β-sheet structures although KFE8 aggregates comprise only one of these structures. Using an excitonic Hamiltonian to simulate the vibrational spectra of model β-sheets, we determine that the spectra are consistent with antiparallel β-sheets with different strand alignments, specifically a two-residue shift in the register of the β-strands. These findings bring forth new insights into how N-terminal acetylation may subtly impact secondary structure, leading to larger effects on overall aggregate morphology. In addition, these results highlight the importance of understanding the residue-level structural differences that result from changes in peptide sequence to facilitate the rational design of peptide materials.     

Amyloid character of self-assembling proteins based on adenovirus fiber shaft sequences

Fibrous proteins found in natural materials such as silk fibroins, spider silks, and viral spikes increasingly serve as a source of inspiration for the design of novel, artificial fibrous materials. The fiber protein from the adenovirus has previously served as a model for the design of artificial, self-assembling fibers. The fibrous shaft of this protein consists of 15-amino-acid sequence repeats that fold into a triple β-spiral motif in their native context. Recombinant proteins based on multimers of simplified consensus shaft repeats were previously reported to form self-assembling fibrils from which filaments could be spun. Here, we describe the structural characterization of these fibrils; X-ray fiber diffraction, Raman spectroscopy, and Congo Red binding strongly suggest an amyloid-type structure for these fibrils, with β-strands arranged perpendicular to the fibril axis. This amyloid structure is distinct from the native β-spiral fold, and similar to amyloid structures formed by short, synthetic peptides corresponding to shaft sequences. We discuss implications for the rational design of novel fibrous materials, based on crystal structure information and knowledge of folding and assembly pathways of natural fibrous proteins.