Self-assembly Of Polypeptides Research Articles

Solid State Self-Assembly Mechanism of RADA16-I Designer Peptide

We report that synthetic RADA16-I peptide transforms to β-strand secondary structure and develops intermolecular organization into β-sheets when stored in the solid state at room temperature. Secondary structural changes were probed using solid state nuclear magnetic resonance spectroscopy (ssNMR) and Fourier transform infrared spectroscopy (FTIR). Intermolecular organization was analyzed via wide-angle X-ray diffraction (WAXD). Observed changes in molecular structure and organization occurred on the time scale of weeks during sample storage at room temperature. We observed structural changes on faster time scales by heating samples above room temperature or by addition of water. Analysis of hydration effects indicates that water can enhance the ability of the peptide to convert to β-strand secondary structure and assemble into β-sheets. However, temperature dependent FTIR and time dependent WAXD data indicate that bound water may hinder the assembly of β-strands into β-sheets. We suggest that secondary structural transformation and intermolecular organization together produce a water-insoluble state. These results reveal insights into the role of water in self-assembly of polypeptides with hydrophilic side chains, and have implications on future optimization of RADA16-I nanofiber production.

Effect of Polypeptide Sequence on Polypeptide Self-Assembly

This study represents a unique example where the self-assembly of five amphiphilic polypeptides was monitored as a function of their hydrophilic-to-lipophilic balance (HLB). The HLB was tuned by preparing a series of polypeptides with aspartic acid (Asp) and phenylalanine (Phe) according to the sequence (Asp(x)Phe(y)) where x and y vary from 1 to 3. The self-assembly of the (Asp(x)Phe(y))(n) polypeptides with M(w) values ranging from 8 to 12 K was studied in solution by fluorescence quenching and nonradiative energy transfer (NRET) fluorescence experiments. Dynamic (DLS) and static (SLS) light scattering studies were used to complement the results obtained. The fluorescence quenching experiments conducted with the chromophore pyrene used as a free probe, physically bound to the polypeptides via hydrophobic interactions, demonstrated that the chromophore was less exposed to the solution for the sequences having a higher hydrophobic character. Protective quenching was also observed for those polypeptides randomly labeled with pyrene where the phenylalanine content was high, whereas pyrene was found to be fully exposed to the quencher for the polypeptides having more hydrophilic sequences. NRET used to probe interpolymeric association was not observed between a naphthalene and a pyrene labeled polypeptide for the polypeptide sequences which were richer in aspartic acid, whereas energy transfer took place with the more hydrophobic polypeptides. This observation led to the conclusion that those sequences with a higher content of aspartic acid essentially generate unimolecular polymeric micelles, whereas those with a higher content of phenylalanine generate polymeric aggregates which offer protection from the solvent to their hydrophobic cargo. The presence of these polymeric aggregates was also confirmed by DLS and SLS studies which suggest that species 100-200 nm in diameter are present in solution.

Compressibility of insulin amyloid fibrils determined by X-ray diffraction in a diamond anvil cell

Amyloid fibrils are fibrous structures that originate from the self-assembly of polypeptides. Their formation is linked to debilitating diseases associated with protein misfolding, including Alzheimer's disease and type-II diabetes. In recent years, it has been suggested that such protein and polypeptide fibrils might provide useful novel nanomaterials. Here, we present the results of a study on the high pressure stability and compressibility of mature amyloid fibrils of insulin by synchrotron X-ray diffraction in a diamond anvil cell. The diffraction results allow a direct estimation of the elastic modulus and the corresponding compression of the cross-β structure along the fiber axis. The average hydrogen bond compressibility is comparable to that in native proteins, suggesting that the fibrils are well-packed.

Self-assembly of polypeptides into left-handedly twisted fibril-like structures

In this paper, we investigated the spontaneous formation of aggregation structures of amyloid-forming peptide (GGVVIA) using a coarse-grained model and Monte Carlo simulations. The effects of concentration and temperature on the formation of different aggregation structures were studied. Three types of aggregation structures, single-layer beta sheet, amorphous beta-sheet aggregate, and fibril-like structures, were observed in our simulations. The fibril-like structures obtained in simulations have a common cross-beta spine structure in which beta sheets twist in a left-handed fashion. The averaged twisting angle of the beta sheet in the fibril-like structures is 12 degrees +/-2 degrees. Moreover, it was found that the peptides in the same beta sheets prefer to arrange in a parallel way, which is consistent with the corresponding GGVVIA crystalline structure. On the other hand, it was found that there is a rich family of beta-sheet stacking patterns in the fibril-like structures suggesting that the fibril structures are more complex than the corresponding crystalline structure and there exist many local free-energy minima rather than a distinct global minimum.

One-dimensional gold nanoparticle arrays by electrostatically directed organization using polypeptide self-assembly.

The study of interactions between particles organized in a linear configuration is interesting from a quantum mechanical perspective, and the anisotropic properties of linear assemblies is of potential interest for the development of solid-state devices.[1– 3 ] This anisotropy may be manifested as a difference in the magnetization and coercivity obtained in a magneticnanoparticle array when a field is applied along the chain or orthogonal to it.[4, 5 ] Nonlinear electrical characteristics[3] or dichroism in the optical spectra with longitudinal and transverse polarizations of light[6, 7 ] in metal nanoparticle arrays are other examples of such anisotropy, and the construction of such arrays would offer opportunities in multiple applications. Engineering matter at submicron length scales has been an area largely dominated by top-down methodologies. Control of interparticle spacing in metal-nanoparticle arrays by using techniques such as electron beam lithography has been found to have a dramatic impact on the optical response of the nanoparticle assembly,[8– 10 ] and has implications in fields such as plasmonics[11] and energy transport.[12, 13 ] The use of templates such as carbon nanotubes [14] and linear pores[15] to construct one-dimensional nanostructures has been demonstrated previously. In addition, polymers have recently begun to play an increasingly active role as elements that can reproducibly direct the arrangement of nanoparticles into functional geometries.[16, 17 ] Self assembling systems offer convenient yet powerful bottom–up strategies for the creation of nanostructures that can be deployed in the realization of functional nanoscale devices.[18, 19 ] The utilization of biomolecules such as DNA,[3, 20 – 24 ] and protein-based materials[25– 27 ] or viruses[28, 29 ] to dictate the organization of nanoparticles has been shown to be an effective and robust paradigm. The use of peptide-based structures[30, 31 ] in nanotechnology confers numerous advantages such as the specification of assembled nanostructure by changes in the primary sequence of the peptide, as well as the adoption of various hierarchical morphologies

Possibilities for 'smart' materials exploiting the self-assembly of polypeptides into fibrils.

Programmed assembly and self-assembly of soft materials offers significant promise for the generation of new types of materials with useful properties. Through evolutionary processes occurring over billions of years, nature has produced numerous optimised building blocks for the controlled assembly of a wide range of complex architectures. Our challenge now is to imitate these naturally occurring processes for technological applications, either using biological molecules such as DNA and proteins, or macromolecular mimics that retain many of the important features of biological molecules while introducing new functionalities. We focus on a single example of biomolecular self-assembly-the self-assembly of polypeptides, including polypeptide mimics, into quasi-one-dimensional fibres-to provide a flavour of the utility of soft biological materials for construction purposes.

Temperature Triggered Self-Assembly of Polypeptides into Multivalent Spherical Micelles

We report herein thermally responsive elastin-like polypeptides (ELPs) in a linear AB diblock architecture with an N-terminal peptide ligand that self-assemble into spherical micelles when heated slightly above body temperature. A series of 10 ELP block copolymers (ELP(BC)'s ) with different molecular weights and hydrophilic-to-hydrophobic block ratios were genetically synthesized by recursive directional ligation. The self-assembly of these polymers from unimers into micelles was investigated by light scattering, fluorescence spectroscopy, and cryo-TEM. These ELP(BC)'s undergo two phase transitions as a function of solution temperature: a unimer-to-spherical micelle transition at an intermediate temperature and a micelle-to-bulk aggregate transition at a higher temperature when the hydrophilic-to-hydrophobic block ratio is between 1:2 and 2:1. The critical micelle temperature is controlled by the length of the hydrophobic block, and the size of the micelle is controlled by both the total ELP(BC) length and hydrophilic-to-hydrophobic block ratio. These polypeptide micelles display a critical micelle concentration in the range 4-8 microM demonstrating the high stability of these structures. These studies have also identified a subset of ELP(BC)'s bearing terminal peptide ligands that are capable of forming multivalent spherical micelles that present multiple copies of the ligand on their corona in the clinically relevant temperature range 37-42 degrees C and target cancer cells. These ELP(BC)'s may be useful for drug targeting by thermally triggered multivalency. More broadly, the design rules uncovered by this study should be applicable to the design of other thermally reversible nanoparticles for diverse applications in medicine and biology.

Charge transport and intrinsic fluorescence in amyloid-like fibrils

The self-assembly of polypeptides into stable, conductive, and intrinsically fluorescent biomolecular nanowires is reported. We have studied the morphology and electrical conduction of fibrils made of an elastin-related polypeptide, poly(ValGlyGlyLeuGly). These amyloid-like nanofibrils, with a diameter ranging from 20 to 250 nm, result from self-assembly in aqueous solution at neutral pH. Their morphological properties and conductivity have been investigated by atomic force microscopy, scanning tunneling microscopy, and two-terminal transport experiments at the micro- and nanoscales. We demonstrate that the nanofibrils can sustain significant electrical conduction in the solid state at ambient conditions and have remarkable stability. We also show intrinsic blue-green fluorescence of the nanofibrils by confocal microscopy analyses. These results indicate that direct (label-free) excitation can be used to investigate the aggregation state or the polymorphism of amyloid-like fibrils (and possibly of other proteinaceous material) and open up interesting perspectives for the use of peptide-based nanowire structures, with tunable physical and chemical properties, for a wide range of nanobiotechnological and bioelectronic applications.

Production of Apoferritin‐Based Bioinorganic Hybrid Nanoparticles by Bacterial Fermentation Followed by Self‐Assembly

Simply made hybrids: Biological, bifunctional nanoparticles (such as that pictured) can be produced by just cultivating Escherichia coli bacteria. No chromatography is required, as isolation and purification are carried out by centrifugation followed by self-assembly of polypeptides to nanoparticles by changing the pH. The particles can be used as a label in bioaffinity assays for biotinylated molecules.

Switch‐peptides as folding precursors in self‐assembling peptides and amyloid fibrillogenesis

The study of conformational transitions of peptides has obtained considerable attention recently because of their importance as a molecular key event in a variety of degenerative diseases. However, the study of peptide self-assembly into beta-sheets and amyloid beta (Abeta) fibrils is strongly hampered by their difficult synthetic access and low solubility. We have recently developed a new concept termed switch-peptides that allows the controlled onset of polypeptide folding and misfolding at physiologic conditions. As a major feature, the folding process is initiated by chemically or enzyme triggered O,N-acyl migration in flexible and soluble folding precursors containing Ser- or Thr-derived switch (S)-elements. The elaborated methodologies are exemplified for the in situ conversion of NPY- and Cyclosporine A-derived prodrugs, as well as for the onset and reversal of alpha and beta conformational transitions in Abeta peptides. In combining orthogonally addressable switch-elements, the consecutive switching on of S-elements gives new insights into the role of individual peptide segments (hot spots) in early processes of polypeptide self-assembly and fibrillogenesis. Finally, the well-known secondary structure disrupting effect of pseudoprolines (PsiPro) is explored for its use as a building block (S-element) in switch-peptides. To this end, synthetic strategies are described, allowing for the preparation of PsiPro-containing folding precursors, exhibiting flexible random-coil conformations devoid of fibril forming propensity. The onset of beta-sheet and fibril formation by restoring the native peptide chain in a single step classify PsiPro-units as the most powerful tool for inhibiting peptide self-assembly, and complement the present methodologies of the switch-concept for the study of fibrillogenesis.

Annealing Prion Protein Amyloid Fibrils at High Temperature Results in Extension of a Proteinase K-resistant Core

Amyloids are highly ordered, rigid beta-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the beta-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 degrees C or, with less efficacy, by prolonged incubation at 37 degrees C at pH 7.5 and is referred to here as "annealing." Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures.

Structural Determinants of Cross-linking and Hydrophobic Domains for Self-Assembly of Elastin-like Polypeptides

Elastin is a major structural protein found in large blood vessels, lung, ligaments, and skin, imparting the physical properties of extensibility and elastic recoil to these tissues. To achieve the required structural durability of the elastic matrix, the elastin monomer, tropoelastin, undergoes ordered assembly into a covalently cross-linked, fibrillar polymeric structure. Human tropoelastin consists of 34 exons coding for alternating hydrophobic and cross-linking domains. Using a series of well-defined recombinant polypeptides based on human elastin sequences mimicking native elastin, we have previously investigated the role of sequence and context of hydrophobic domains in elastin self-assembly. Here, we demonstrate that the structure of both cross-linking and hydrophobic domains have significant effects on the assembly of these polypeptides. Removing a putative flexible hinge region in the center of a cross-linking domain substantially increased the alpha-helical content and strongly promoted their self-aggregation. However, while trifluoroethanol (TFE) promoted and urea inhibited self-assembly of these polypeptides, these effects were not predominantly due to altered alpha-helicity of the polypeptides. Our results suggest that, while increased alpha helicity also favors this process, the major effect of TFE to promote organized self-assembly of elastin-like polypeptides is likely related to direct effects of this cosolvent on hydrophobic domains. Such simple elastin polypeptide models can provide an important tool for understanding the relationships between sequence, structure, and polymeric assembly of elastin.

Microarrays of Peptide Fibrils Created by Electrostatically Controlled Deposition

The adsorption of β-sheet-rich fibrils produced in vitro via the self-assembly of polypeptides onto surfaces is found to be governed by electrostatic interactions. Chemically patterned substrates are used to create lateral micropatterns of fibrils and hybrid microstructures of fibrils with inorganic nanoparticles (see Figure). β-sheet-rich fibrils are promising as functional biomaterials.

Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1

Bones and teeth are biocomposites that require controlled mineral deposition during their self-assembly to form tissues with unique mechanical properties. Acidic extracellular matrix proteins play a pivotal role during biomineral formation. However, the mechanisms of protein-mediated mineral initiation are far from understood. Here we report that dentin matrix protein 1 (DMP1), an acidic protein, can nucleate the formation of hydroxyapatite in vitro in a multistep process that begins by DMP1 binding calcium ions and initiating mineral deposition. The nucleated amorphous calcium phosphate precipitates ripen and nanocrystals form. Subsequently, these expand and coalesce into microscale crystals elongated in the c-axis direction. Characterization of the functional domains in DMP1 demonstrated that intermolecular assembly of acidic clusters into a beta-sheet template was essential for the observed mineral nucleation. Protein-mediated initiation of nanocrystals, as discussed here, might provide a new methodology for constructing nanoscale composites by self-assembly of polypeptides with tailor-made peptide sequences.