Nanoscale Heterogeneity of the Molecular Structure of Individual hIAPP Amyloid Fibrils Revealed with Tip‐Enhanced Raman Spectroscopy

Type 2 diabetes mellitus is characterized by the pathological deposition of fibrillized protein, known as amyloids. It is thought that oligomers and/or amyloid fibrils formed from human islet amyloid polypeptide (hIAPP or amylin) cause cell death by membrane damage. The molecular structure of hIAPP amyloid fibrils is dominated by β‐sheet structure, as probed with conventional infrared and Raman vibrational spectroscopy. However, with these techniques it is not possible to distinguish between the core and the surface structure of the fibrils. Since the fibril surface crucially affects amyloid toxicity, it is essential to know its structure. Here the surface molecular structure and amino acid residue composition of hIAPP fibrils are specifically probed with nanoscale resolution using tip‐enhanced Raman spectroscopy (TERS). The fibril surface mainly contains unordered or α‐helical structures, in contrast to the β‐sheet‐rich core. This experimentally validates recent models of hIAPP amyloids based on NMR measurements. Spatial mapping of the surface structure reveals a highly heterogeneous surface structure. Finally, TERS can probe fibrils formed on a lipid interface, which is more representative of amyloids in vivo.     

Thioflavin T‐Amyloid Hybrid Nanostructure for Biocatalytic Photosynthesis

Amyloidogenic peptides can self‐assemble into highly ordered nanostructures consisting of cross β‐sheet‐rich networks that exhibit unique physicochemical properties and high stability. Light‐harvesting amyloid nanofibrils are constructed by employing insulin as a building block and thioflavin T (ThT) as a amyloid‐specific photosensitizer. The ability of the self‐assembled amyloid scaffold to accommodate and align ThT in high density on its surface allows for efficient energy transfer from the chromophores to the catalytic units in a similar way to natural photosystems. Insulin nanofibrils significantly enhance the photoactivity of ThT by inhibiting nonradiative conformational relaxation around the central C? C bonds and narrowing the distance between ThT molecules that are bound to the β‐sheet‐rich amyloid structure. It is demonstrated that the ThT‐amyloid hybrid nanostructure is suitable for biocatalytic solar‐to‐chemical conversion by integrating the light‐harvesting amyloid module (for nicotinamide cofactor regeneration) with a redox biocatalytic module (for enzymatic reduction).     

Carbon Nanodot‐Sensitized Modulation of Alzheimer's β‐Amyloid Self‐Assembly,Disassembly, and Toxicity

The self‐assembly of amyloidogenic peptides into β‐sheet‐rich aggregates is a general feature of many neurodegenerative diseases, including Alzheimer's disease, which signifies the need for the effective attenuation of amyloid aggregation toward alleviating amyloid‐associated neurotoxicity. This study reports that photoluminescent carbon nanodots (CDs) can effectively suppress Alzheimer's β‐amyloid (Aβ) self‐assembly and function as a β‐sheet breaker disintegrating preformed Aβ aggregates. This study synthesizes CDs using ammonium citrate through one‐pot hydrothermal treatment and passivates their surface with branched polyethylenimine (bPEI). The bPEI‐coated CDs (bPEI@CDs) exhibit hydrophilic and cationic surface characteristics, which interact with the negatively charged residues of Aβ peptides, suppressing the aggregation of Aβ peptides. Under light illumination, bPEI@CDs display a more pronounced effect on Aβ aggregation and on the dissociation of β‐sheet‐rich assemblies through the generation of reactive oxygen species from photoactivated bPEI@CDs. The light‐triggered attenuation effect of Aβ aggregation using a series of experiments, including photochemical and microscopic analysis, is verified. Furthermore, the cell viability test confirms the ability of photoactivated bPEI@CDs for the suppression of Aβ‐mediated cytotoxicity, indicating bPEI@CDs' potency as an effective anti‐Aβ neurotoxin agent.     

Amyloid Fibrils as Building Blocks for Natural and Artificial Functional Materials

Proteinaceous materials based on the amyloid core structure have recently been discovered at the origin of biological functionality in a remarkably diverse set of roles, and attention is increasingly turning towards such structures as the basis of artificial self‐assembling materials. These roles contrast markedly with the original picture of amyloid fibrils as inherently pathological structures. Here we outline the salient features of this class of functional materials, both in the context of the functional roles that have been revealed for amyloid fibrils in nature, as well as in relation to their potential as artificial materials. We discuss how amyloid materials exemplify the emergence of function from protein self‐assembly at multiple length scales. We focus on the connections between mesoscale structure and material function, and demonstrate how the natural examples of functional amyloids illuminate the potential applications for future artificial protein based materials.     

Spatially Controlled Amyloid Reactions Using Organic Electronics

Abnormal protein aggregates, so called amyloid fibrils, are mainly known as pathological hallmarks of a wide range of diseases, but in addition these robust well‐ordered self‐assembled natural nanostructures can also be utilized for creating distinct nanomaterials for bioelectronic devices. However, current methods for producing amyloid fibrils in vitro offer no spatial control. Herein, we demonstrate a new way to produce and spatially control the assembly of amyloid‐like structures using an organic electronic ion pump (OEIP) to pump distinct cations to a reservoir containing a negatively charged polypeptide. The morphology and kinetics of the created proteinaceous nanomaterials depends on the ion and current used, which we leveraged to create layers incorporating different conjugated thiophene derivatives, one fluorescent (p‐FTAA) and one conducting (PEDOT‐S). We anticipate that this new application for the OEIP will be useful for both biological studies of amyloid assembly and fibrillogenesis as well as for creating new bioelectronic nanomaterials and devices.     

Amyloid Self‐Assembly of hIAPP8‐20 via the Accumulation of Helical Oligomers, α‐Helix to β‐Sheet Transition,and Formation of β‐Barrel Intermediates

The self‐assembly of human islet amyloid polypeptide (hIAPP) into β‐sheet‐rich nanofibrils is associated with the pathogeny of type 2 diabetes. Soluble hIAPP is intrinsically disordered with N‐terminal residues 8–17 as α‐helices. To understand the contribution of the N‐terminal helix to the aggregation of full‐length hIAPP, here the oligomerization dynamics of the hIAPP fragment 8–20 (hIAPP8‐20) are investigated with combined computational and experimental approaches. hIAPP8‐20 forms cross‐β nanofibrils in silico from isolated helical monomers via the helical oligomers and α‐helices to β‐sheets transition, as confirmed by transmission electron microscopy, atomic force microscopy, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and reversed‐phase high performance liquid chromatography. The computational results also suggest that the critical nucleus of aggregation corresponds to hexamers, consistent with a recent mass‐spectroscopy study of hIAPP8‐20 aggregation. hIAPP8‐20 oligomers smaller than hexamers are helical and unstable, while the α‐to‐β transition starts from the hexamers. Converted β‐sheet‐rich oligomers first form β‐barrel structures as intermediates before aggregating into cross‐β nanofibrils. This study uncovers a complete picture of hIAPP8‐20 peptide oligomerization, aggregation nucleation via conformational conversion, formation of β‐barrel intermediates, and assembly of cross‐β protofibrils, thereby shedding light on the aggregation of full‐length hIAPP, a hallmark of pancreatic beta‐cell degeneration.     

Beta‐Sheet‐Forming,Self‐Assembled Peptide Nanomaterials towards Optical,Energy, and Healthcare Applications

Peptide self‐assembly is an attractive route for the synthesis of intricate organic nanostructures that possess remarkable structural variety and biocompatibility. Recent studies on peptide‐based, self‐assembled materials have expanded beyond the construction of high‐order architectures; they are now reporting new functional materials that have application in the emerging fields such as artificial photosynthesis and rechargeable batteries. Nevertheless, there have been few reviews particularly concentrating on such versatile, emerging applications. Herein, recent advances in the synthesis of self‐assembled peptide nanomaterials (e.g., cross β‐sheet‐based amyloid nanostructures, peptide amphiphiles) are selectively reviewed and their new applications in diverse, interdisciplinary fields are described, ranging from optics and energy storage/conversion to healthcare. The applications of peptide‐based self‐assembled materials in unconventional fields are also highlighted, such as photoluminescent peptide nanostructures, artificial photosynthetic peptide nanomaterials, and lithium‐ion battery components. The relation of such functional materials to the rapidly progressing biomedical applications of peptide self‐assembly, which include biosensors/chips and regenerative medicine, are discussed. The combination of strategies shown in these applications would further promote the discovery of novel, functional, small materials.     

Nanoparticle‐Induced Folding and Fibril Formation of Coiled‐Coil‐Based Model Peptides

Nanomedicine is a rapidly growing field that has the potential to deliver treatments for many illnesses. However, relatively little is known about the biological risks of nanoparticles. Some studies have shown that nanoparticles can have an impact on the aggregation properties of proteins, including fibril formation. Moreover, these studies also show that the capacity of nanoscale objects to induce or prevent misfolding of the proteins strongly depends on the primary structure of the protein. Herein, light is shed on the role of the peptide primary structure in directing nanoparticle‐induced misfolding by means of two model peptides. The design of these peptides is based on the α‐helical coiled‐coil folding motif, but also includes features that enable them to respond to pH changes, thus allowing pH‐dependent β‐sheet formation. Previous studies showed that the two peptides differ in the pH range required for β‐sheet folding. Time‐dependent circular dichroism spectroscopy and transmission electron microscopy are used to characterize peptide folding and aggregate morphology in the presence of negatively charged gold nanoparticles (AuNPs). Both peptides are found to undergo nanoparticle‐induced fibril formation. The determination of binding parameters by isothermal titration calorimetry further reveals that the different propensities of both peptides to form amyloid‐like structures in the presence of AuNPs is primarily due to the binding stoichiometry to the AuNPs. Modification of one of the peptide sequences shows that AuNP‐induced β‐sheet formation is related to the structural propensity of the primary structure and is not a generic feature of peptide sequences with a sufficiently high binding stoichiometry to the nanoparticles.     

Interface‐Directed Self‐Assembly of Cell‐Laden Microgels

Cell‐laden hydrogels show great promise for creating engineered tissues. However, a major shortcoming with these systems has been the inability to fabricate structures with controlled micrometer‐scale features on a biologically relevant length scale. In this Full Paper, a rapid method is demonstrated for creating centimeter‐scale, cell‐laden hydrogels through the assembly of shape‐controlled microgels or a liquid–air interface. Cell‐laden microgels of specific shapes are randomly placed on the surface of a high‐density, hydrophobic solution, induced to aggregate and then crosslinked into macroscale tissue‐like structures. The resulting assemblies are cell‐laden hydrogel sheets consisting of tightly packed, ordered microgel units. In addition, a hierarchical approach creates complex multigel building blocks, which are then assembled into tissues with precise spatial control over the cell distribution. The results demonstrate that forces at an air–liquid interface can be used to self‐assemble spatially controllable, cocultured tissue‐like structures.     

Dual‐pH Sensitive Charge‐Reversal Polypeptide Micelles for Tumor‐Triggered Targeting Uptake and Nuclear Drug Delivery

A novel dual‐pH sensitive charge‐reversal strategy is designed to deliver antitumor drugs targeting to tumor cells and to further promote the nuclei internalization by a stepwise response to the mildly acidic extracellular pH (≈6.5) of a tumor and endo/lysosome pH (≈5.0). Poly(l ‐lysine)‐block–poly(l ‐leucine) diblock copolymer is synthesized and the lysine amino residues are amidated by 2,3‐dimethylmaleic anhydride to form β‐carboxylic amide, making the polypeptides self‐assemble into negatively charged micelles. The amide can be hydrolyzed when exposed to the mildly acidic tumor extracellular environment, which makes the micelles switch to positively charged and they are then readily internalized by tumor cells. A nuclear targeting Tat peptide is further conjugated to the polypeptide via a click reaction. The Tat is amidated by succinyl chloride to mask its positive charge and cell‐penetrating function and thus to inhibit nonspecific cellular uptake. After the nanoparticles are internalized into the more acidic intracellular endo/lysosomes, the Tat succinyl amide is hydrolyzed to reactivate the Tat nuclear targeting function, promoting nanoparticle delivery into cell nuclei. This polypeptide nanocarrier facilitates tumor targeting and nuclear delivery simultaneously by simply modifying the lysine amino residues of polylysine and Tat into two different pH‐sensitive β‐carboxylic amides.     

Diverse Morphologies of Self‐Assemblies from Homoditopic 1,18‐Nucleotide‐Appended Bolaamphiphiles: Effects of Nucleobases and Complementary Oligonucleotides

The synthesis of 1,18‐nucleotide‐appended bolaamphiphiles (1 , 2 , 4 , and 6) is reported, in which a 3′‐phosphorylated guanidine, adenosine, thymidine, or cytidine is connected to each end of an octadecamethylene chain. Single‐component self‐assemblies and binary self‐assemblies with the complementary oligonucleotides dC ₂₀ , dT ₂₀ , dA ₂₀ , and dG ₂₀ are studied by atomic force microscopy, powder X‐ray diffraction analysis, temperature‐dependent UV absorption, circular dichroism, and attenuated total‐reflection Fourier‐transform infrared spectroscopy. The single‐component self‐assembly of 1 forms a two‐dimensional sheet, whereas the binary self‐assembly 1 / dC ₂₀ gives helical nanofibers. Non‐helical nanofibers are observed for the single‐component self‐assemblies of 2 and 4 , and helical nanofibers form from the binary self‐assembly 2 / dT ₂₀ . Interestingly, helical nanorod structures are obtained from the binary self‐assembly 4 / dA ₂₀ , and the aligned nanorods form a nematic phase. The single‐component and binary self‐assemblies from 6 give unilamellar vesicles owing to a lack of stacking interaction between the cytosine moieties.     

Electrical Conductivity,Selective Adhesion,and Biocompatibility in Bacteria‐Inspired Peptide–Metal Self‐Supporting Nanocomposites

Bacterial type IV pili (T4P) are polymeric protein nanofibers that have diverse biological roles. Their unique physicochemical properties mark them as a candidate biomaterial for various applications, yet difficulties in producing native T4P hinder their utilization. Recent effort to mimic the T4P of the metal‐reducing Geobacter sulfurreducens bacterium led to the design of synthetic peptide building blocks, which self‐assemble into T4P‐like nanofibers. Here, it is reported that the T4P‐like peptide nanofibers efficiently bind metal oxide particles and reduce Au ions analogously to their native counterparts, and thus give rise to versatile and multifunctional peptide–metal nanocomposites. Focusing on the interaction with Au ions, a combination of experimental and computational methods provides mechanistic insight into the formation of an exceptionally dense Au nanoparticle (AuNP) decoration of the nanofibers. Characterization of the thus‐formed peptide–AuNPs nanocomposite reveals enhanced thermal stability, electrical conductivity from the single‐fiber level up, and substrate‐selective adhesion. Exploring its potential applications, it is demonstrated that the peptide–AuNPs nanocomposite can act as a reusable catalytic coating or form self‐supporting immersible films of desired shapes. The films scaffold the assembly of cardiac cells into synchronized patches, and present static charge detection capabilities at the macroscale. The study presents a novel T4P‐inspired biometallic material.     

Hybrid Nanoscopy of Hybrid Nanomaterials

The combination of complementary techniques to characterize materials at the nanoscale is crucial to gain a more complete picture of their structure, a key step to design and fabricate new materials with improved properties and diverse functions. Here it is shown that correlative atomic force microscopy (AFM) and localization‐based super‐resolution microscopy is a useful tool that provides insight into the structure and emissive properties of fluorescent β‐lactoglobulin (βLG) amyloid‐like fibrils. These hybrid materials are made by functionalization of βLG with organic fluorophores and quantum dots, the latter being relevant for the production of 1D inorganic nanostructures templated by self‐assembling peptides. Simultaneous functionalization of βLG fibers by QD655 and QD525 allows for correlative AFM and two‐color super‐resolution fluorescence imaging of these hybrid materials. These experiments allow the combination of information about the topography and number of filaments that compose a fibril, as well as the emissive properties and nanoscale spatial distribution of the attached fluorophores. This study represents an important step forward in the characterization of multifunctionalized hybrid materials, a key challenge in nanoscience.     

Coaggregation of κ‐Casein and β‐Lactoglobulin Produces Morphologically Distinct Amyloid Fibrils

The unfolding, misfolding, and aggregation of proteins lead to a variety of structural species. One form is the amyloid fibril, a highly aligned, stable, nanofibrillar structure composed of β‐sheets running perpendicular to the fibril axis. β‐Lactoglobulin (β‐Lg) and κ‐casein (κ‐CN) are two milk proteins that not only individually form amyloid fibrillar aggregates, but can also coaggregate under environmental stress conditions such as elevated temperature. The aggregation between β‐Lg and κ‐CN is proposed to proceed via disulfide bond formation leading to amorphous aggregates, although the exact mechanism is not known. Herein, using a range of biophysical techniques, it is shown that β‐Lg and κ‐CN coaggregate to form morphologically distinct co‐amyloid fibrillar structures, a phenomenon previously limited to protein isoforms from different species or different peptide sequences from an individual protein. A new mechanism of aggregation is proposed whereby β‐Lg and κ‐CN not only form disulfide‐linked aggregates, but also amyloid fibrillar coaggregates. The coaggregation of two structurally unrelated proteins into cofibrils suggests that the mechanism can be a generic feature of protein aggregation as long as the prerequisites for sequence similarity are met.     

Directed Assembly of Gold Nanorods by Terminal‐Base Pairing of Surface‐Grafted DNA

Directed assemblies of anisotropic metal nanoparticles exhibit attractive physical and chemical properties. However, an effective methodology to prepare differently directed assemblies from the same anisotropic nanoparticles is not yet available. Gold nanorods (AuNRs) region‐selectively modified with different DNA strands can form side‐by‐side (SBS) and end‐to‐end (ETE) assemblies in a non‐crosslinking manner. When the complementary DNA is hybridized to the surface‐bound DNA, stacking interaction between the blunt ends takes place in the designated regions. Such AuNRs assemble into highly ordered structures, assisted by capillary forces emerging on the substrate surface. Moreover, insertion of a mercury(II)‐mediated thymine–thymine base pair into the periphery of the DNA layer allows selective formation of the SBS or ETE assemblies from the strictly identical AuNRs with or without mercury(II).     

Switchable Helical Structures Formed by the Hierarchical Self‐Assembly of Laterally Tethered Nanorods

The formation of helical scrolls formed by self‐assembly of tethered nanorod amphiphiles and their molecular analogs are investigated. A model bilayer sheet assembled by laterally tethered nanorods is simulated and shown that it can fold into distinct helical morphologies under different solvent conditions. The helices can reversibly transform from one morphology to another by dynamically changing the solvent condition. This model serves both to inspire the fabrication of laterally tethered nanorods for assembling helices at nanometer scales and as a proof‐of‐concept for engineering switchable nanomaterials via hierarchical self‐assembly.     

Bioinspired Stable and Photoluminescent Assemblies for Power Generation

Peptide assemblies are ideal components for eco‐friendly optoelectronic energy harvesting devices due to their intrinsic biocompatibility, ease of fabrication, and flexible functionalization. However, to date, their practical applications have been limited due to the difficulty in obtaining stable, high‐performance devices. Here, it is shown that the tryptophan‐based simplest peptide cyclo‐glycine‐tryptophan (cyclo‐GW) forms mechanically robust (elastic modulus up to 24.0 GPa) and thermally stable up to 370 °C monoclinic crystals, due to a supramolecular packing combining dense parallel β‐sheet hydrogen bonding and herringbone edge‐to‐face aromatic interactions. The directional and extensive driving forces further confer unique optical properties, including aggregation‐induced blue emission and unusual stable photoluminescence. Moreover, the crystals produce a high and sustained open‐circuit voltage (1.2 V) due to a high piezoelectric coefficient of 14.1 pC N^?1. These findings demonstrate the feasibility of utilizing self‐assembling peptides for fabrication of biointegrated microdevices that combine high structural stability, tailored optoelectronics, and significant energy harvesting properties.     

An “On‐Site Transformation” Strategy for Treatment of Bacterial Infection

To date, numerous nanosystems have been developed as antibiotic replacements for bacterial infection treatment. However, these advanced systems are limited owing to their nontargeting accumulation and the consequent side effects. Herein, transformable polymer–peptide biomaterials have been developed that enable specific accumulation in the infectious site and long‐term retention, resulting in enhanced binding capability and killing efficacy toward bacteria. The polymer–peptide conjugates are composed of a chitosan backbone and two functional peptides, i.e., an antimicrobial peptide and a poly(ethylene glycol)‐tethered enzyme‐cleavable peptide (CPC‐1). The CPC‐1 initially self‐assembles into nanoparticles with pegylated coronas. Upon the peptides are cleaved by the gelatinase secreted by a broad spectrum of bacterial species, the resultant compartments of nanoparticles spontaneously transformed into fibrous nanostructures that are stabilized by enhanced chain–chain interaction, leading to exposure of antimicrobial peptide residues for multivalent cooperative electrostatic interactions with bacterial membranes. Intriguingly, the in situ morphological transformation also critically improves the accumulation and retention of CPC‐1 in infectious sites in vivo, which exhibits highly efficient antibacterial activity. This proof‐of‐concept study demonstrates that pathological environment‐driven smart self‐assemblies may provide a new idea for design of high‐performance biomaterials for disease diagnostics and therapeutics.     

Peptide Nanophotonics: From Optical Waveguiding to Precise Medicine and Multifunctional Biochips

Optical waveguiding phenomena found in bioinspired chemically synthesized peptide nanostructures are a new paradigm which can revolutionize emerging fields of precise medicine and health monitoring. A unique combination of their intrinsic biocompatibility with remarkable multifunctional optical properties and developed nanotechnology of large peptide wafers makes them highly promising for new biomedical light therapy tools and implantable optical biochips. This Review highlights a new field of peptide nanophotonics. It covers peptide nanotechnology and the fabrication process of peptide integrated optical circuits, basic studies of linear and nonlinear optical phenomena in biological and bioinspired nanostructures, and their passive and active optical waveguiding. It is shown that the optical properties of this generation of bio‐optical materials are governed by fundamental biological processes. Refolding the peptide secondary structure is followed by wideband optical absorption and visible tunable fluorescence. In peptide optical waveguides, such a bio‐optical effect leads to switching from passive waveguiding mode in native α‐helical phase to an active one in the β‐sheet phase. The found active waveguiding effect in β‐sheet fiber structures below optical diffraction limit opens an avenue for the future development of new bionanophotonics in ultrathin peptide/protein fibrillar structures toward advanced biomedical nanotechnology.     

Controlling Long‐Distance Photoactuation with Protein Additives

Long‐distance wireless actuation indicates precise remote control over materials, sensors, and devices that are widely utilized in biomedical, defence, disaster relief, deep ocean, and outer space applications to replace human work. Unlike radio frequency (RF) control, which has low tolerance toward electromagnetic interference (EMI), light control represents a promising method to overcome EMI. Nonetheless, long‐distance light‐controlled wireless actuation able to compete with RF control has not been achieved until now due to the lack of highly light‐sensitive actuator designs. Here, it is demonstrate that amyloid‐like protein aggregates can organize photomodule single‐layer reduced graphene oxide (rGO) into a well‐defined multilayer stack to display long‐distance photoactuation. The amyloid‐like proteinaceous component docks the rGO layers together to form a hybrid film, which can reliably adhere onto various material surfaces with robust interfacial adhesion. The sensitive photothermal effect and a fast bending in 1 s to switch a circuit are achieved after forming the film on a plastic substrate and irradiating the bilayer film with a blue laser from 100 m away. A photoactuation distance of 50 km can be further extrapolated based on a commercial high‐power laser. This study reveals the great potential of amyloid‐like aggregates in remote light control of robots and devices.