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。     

From short peptides to nanofibers to macromolecular assemblies in biomedicine

In the last few years, a variety of self-assembling short peptides that consist exclusively of simple amino acids have been designed and modified. These peptides exhibit self-assembling dynamic behaviors. At the molecular structural level, they form α-helical, β-sheet and β-hairpins structures in water. These structures further undergo spontaneous assembly to form nanofibers which aggregate into supramolecular scaffolds that entrap large volumes of water. Furthermore, nanostructures and supramolecular structures that self-organized from these short peptides also have a broad spectrum of biotechnological applications. They are useful as biological materials for 2D and 3D tissue cell cultures, regenerative and reparative medicine, tissue engineering as well as injectable drug delivery matrices that gel in situ. We have endeavored to do a comprehensive review of short peptides that form nanofibrous hydrogels. In particular, we have focused on recent advances in peptide assembly motifs and applications.     

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.     

Aromatic cross-strand ladders control the structure and stability of beta-rich peptide self-assembly mimics

Though β-rich self-assemblies comprise a major structural class of polypeptides, a detailed understanding of the determinants of their structure and stability is lacking. In particular, the roles of repetitive stretches of side chains running the long axis of these β-sheets, termed “cross-strand ladders,” remain poorly characterized due to the inherently insoluble and heterogeneous nature of self-assemblies. To overcome these experimental challenges, we have established a complementary experimental system termed “peptide self-assembly mimics” (PSAMs). The PSAMs capture a defined number of self-assembly-like peptide repeats within a soluble β-rich protein, making structural and energetic studies possible. In this work, we investigated the role of cross-strand ladders containing aromatic residues, which are prominent in self-assembling peptides. A combination of solution data and high-resolution crystal structures revealed that a single cross-strand ladder consisting solely of Tyr significantly stabilized, rigidified, and flattened the PSAM β-sheet. These characteristics would stabilize each β-sheet layer of a self-assembly and direct sheet conformations compatible with lamination. Our results therefore provide a rationale for the abundance of aromatic amino acids in fibril-forming peptides and establish important roles of cross-strand Tyr ladders in the structure and stability of β-rich peptide self-assemblies.     

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.     

High-resolution structure of a self-assembly-competent form of a hydrophobic peptide captured in a soluble beta-sheet scaffold

β-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer β-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile₅), which is insoluble and forms β-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of self-assembly. The 1.1-Å crystal structure revealed that the Ile₅ stretch forms a highly regular β-strand within this flat β-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile₅ peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat β-strand of the Ile₅ stretch primed for self-assembly is a low-energy conformation of the Ile₅ stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to “solubilize” an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides.     

Self-assembly in mesoscopic dimension and artificial supramolecular membranes

The development of mesoscopic supramolecular architectures is an area of growing interest. The field has grown from early works on bilayer membranes, and the design of large (super- or giant-) amphiphiles, hybrid amphiphiles and supramolecular membranes have now been described. Impartment of amphiphilicity to a unit supermolecule allows their hierarchical self-assembly to the mesoscopic structures. A supramolecular combinatorial approach is useful in the development of functional self-assemblies. In addition, self-assembly in ionic liquids has been introduced as a promising area in materials chemistry.     

Self-complementary peptides for the formation of collagen-like triple helical supramolecules

Collagen is acknowledged as one of the most prominent biomaterials on account of its high biocompatibility and biostability. The development of artificial collagens to replace the animal-derived collagens presents a challenge in the formation of safer and highly functionalized biomaterials. Here, a novel peptide-based system for obtaining collagen-like supramolecules via a spontaneous self-assembling process is described. The designed collagen-like peptides are self-complementary trimers in which each of the 24-mer peptide strands is tethered by two cystine knots forming a staggered arrangement. Their self-assembling ability in aqueous solution was analyzed by circular dichroism, ultrafiltration, and laser diffraction particle size estimation. The obtained results indicate that the staggered trimers form large supramolecular architectures through intermolecular triple helix-formation.     

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.     

Gold DNA-conjugates: ion specific self-assembly of gold nanoparticles via the dG-quartet

The Oxytricha telomere DNA hairpin 5'-d(G4T4G4) immobilized on 13 nm gold nanoparticles forms a supramolecular assembly via dGC-quartets, as determined by the color change and by SEM. The aggregation is ion-dependent and selective for sodium ions. K+ is less efficient while Li+ and Cs+ do not drive the aggregation. This work is the first effort exploring the use of secondary structures of DNA (quadruplexes) for producing self-assemblies of gold nanoparticles.     

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.     

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.     

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.     

The structure and protein binding of amyloid-specific dye reagents

The self-assembling tendency and protein complexation capability of dyes related to Congo red and also some dyes of different structure were compared to explain the mechanism of Congo red binding and the reason for its specific affinity for beta-structure. Complexation with proteins was measured directly and expressed as the number of dye molecules bound to heat-aggregated IgG and to two light chains with different structural stability. Binding of dyes to rabbit antibodies was measured indirectly as the enhancement effect of the dye on immune complex formation. Self-assembling was tested using dynamic light scattering to measure the size of the supramolecular assemblies. In general the results show that the supramolecular form of a dye is the main factor determining its complexation capability. Dyes that in their compact supramolecular organization are ribbon-shaped may adhere to polypeptides of beta-conformation due to the architectural compatibility in this unique structural form. The optimal fit in complexation seems to depend on two contradictory factors involving, on the one hand, the compactness of the non-covalently stabilized supramolecular ligand, and the dynamic character producing its plasticity on the other. As a result, the highest protein binding capability is shown by dyes with a moderate self-assembling tendency, while those arranging into either very rigid or very unstable supramolecular entities are less able to bind.     

GOLD DNA-CONJUGATES: ION SPECIFIC SELF-ASSEMBLY OF GOLD NANOPARTICLES VIA THE DG-QUARTET

The Oxytricha telomere DNA hairpin 5′-d(G4T4G4) immobilized on 13 nm gold nanoparticles forms a supramolecular assembly via dG-quartets, as determined by the color change and by SEM. The aggregation is ion-dependent and selective for sodium ions. K⁺ is less efficient while Li⁺ and Cs⁺ do not drive the aggregation. This work is the first effort exploring the use of secondary structures of DNA (quadruplexes) for producing self-assemblies of gold nanoparticles.     

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.     

Ion-specific aggregation of gold-DNA nanoparticles using the dG quartet hairpin 5'-d(G4T4G4)

Ion-selective self-assembly of Au nanoparticles is described. The Oxytricha telomere DNA hairpin 5'-d(G4T4G4) immobilized on 13-nm Au nanoparticles forms a supramolecular assembly via dG-quartets, as determined by the color change and by scanning electron microscopy (SEM). The aggregation is ion-dependent and selective for Na+ ions. K+ is less efficient, while Li+ and Cs+ do not drive the aggregation. This work is the first effort to explore the use of secondary structures of DNA (quadruplexes) for producing self-assemblies of Au nanoparticles. It can be utilized to generate novel devices and materials, potentially useful for sensing and other applications in medicinal or engineering science.     

Atomistic simulation approach to a continuum description of self-assembled beta-sheet filaments

We investigated the supramolecular structure and continuum mechanical properties of a beta-sheet nanofiber comprised of a self-assembling peptide ac-[RARADADA]2-am using computer simulations. The supramolecular structure was determined by constructing candidate filaments with dimensions compatible with those observed in atomic force microscopy and selecting the most stable ones after running molecular dynamics simulations on each of them. Four structures with different backbone hydrogen-bonding patterns were identified to be similarly stable. We then quantified the continuum mechanical properties of these identified structures by running three independent simulations: thermal motion analysis, normal mode analysis, and steered molecular dynamics. Within the range of deformations investigated, the filament showed linear elasticity in transverse directions with an estimated persistence length of 1.2-4.8 microm. Although side-chain interactions govern the propensity and energetics of filament self-assembly, we found that backbone hydrogen-bonding interactions are the primary determinant of filament elasticity, as demonstrated by its effective thickness, which is smaller than that estimated by atomic force microscopy or from the molecular geometry, as well as by the similar bending stiffness of a model filament without charged side chains. The generality of our approach suggests that it should be applicable to developing continuum elastic ribbon models of other beta-sheet filaments and amyloid fibrils.     

Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-D mass spectrometry

Posttranslational glycosylation is critical for biological function of many proteins, but its structural characterization is complicated by natural heterogeneity, multiple glycosylation sites, and different forms. Here, a top-down mass spectrometry (MS) characterization is applied to three constructs of the Fc segment of IgE: Fcepsilon(3-4) (52 kDa) and Fcepsilon(2-3-4)(2) (76 kDa) disulfide-bonded homodimers. Fourier transform MS of a reduced sample of Fcepsilon(2-3-4) gave molecular masses of 37 527, 37 689, 37 851, and 38 014 Da, directly characterizing multiple glycoforms (hexose = 162 Da) without chromatographic separation. Limited proteolysis of the nonreduced Fcepsilon(2-3-4)(2) protein yielded a peptide mixture with molecular weight values that agreed with those expected from the DNA sequence. The single glycosylation site in these constructs was identified, and quantities were determined of five glycoforms that agreed within +/-2% of the molecular ion values. The 2-D mass spectrum of two glycosylated peptides showed these to have high-mannose structures, -GlcNAc-(hex)(n)(), demonstrating that Fcepsilon(2-3-4) has a single such structure of n = 5-9. For a mutated sample of Fcepsilon(3-4), in addition to five glycoforms, MS showed a molecular discrepancy that could be assigned with proteolysis and 2-D mass spectra to the oxidation of two methionines and an additional residue difference.