Engineering Globular Protein Vesicles through Tunable Self‐Assembly of Recombinant Fusion Proteins

Vesicles assembled from folded, globular proteins have potential for functions different from traditional lipid or polymeric vesicles. However, they also present challenges in understanding the assembly process and controlling vesicle properties. From detailed investigation of the assembly behavior of recombinant fusion proteins, this work reports a simple strategy to engineer protein vesicles containing functional, globular domains. This is achieved through tunable self‐assembly of recombinant globular fusion proteins containing leucine zippers and elastin‐like polypeptides. The fusion proteins form complexes in solution via high affinity binding of the zippers, and transition through dynamic coacervates to stable hollow vesicles upon warming. The thermal driving force, which can be tuned by protein concentration or temperature, controls both vesicle size and whether vesicles are single or bi‐layered. These results provide critical information to engineer globular protein vesicles via self‐assembly with desired size and membrane structure.     

Metal Ion‐Induced Self‐Assembly of a Multi‐Responsive Block Copolypeptide into Well‐Defined Nanocapsules

Protein cages are an interesting class of biomaterials with potential applications in bionanotechnology. Therefore, substantial effort is spent on the development of capsule‐forming designer polypeptides with a tailor‐made assembly profile. The expanded assembly profile of a triblock copolypeptide consisting of a metal ion chelating hexahistidine‐tag, a stimulus‐responsive elastin‐like polypeptide block, and a pH‐responsive morphology‐controlling viral capsid protein is presented. The self‐assembly of this multi‐responsive protein‐based block copolymer is triggered by the addition of divalent metal ions. This assembly process yields monodisperse nanocapsules with a 20 nm diameter composed of 60 polypeptides. The well‐defined nanoparticles are the result of the emergent properties of all the blocks of the polypeptide. These results demonstrate the feasibility of hexahistidine‐tags to function as supramolecular cross‐linkers. Furthermore, their potential for the metal ion‐mediated encapsulation of hexahistidine‐tagged proteins is shown.     

Designing Non‐Native Iron‐Binding Site on a Protein Cage for Biological Synthesis of Nanoparticles

In biomineralization processes, a supramolecular organic structure is often used as a template for inorganic nanomaterial synthesis. The E2 protein cage derived from Geobacillus stearothermophilus pyruvate dehydrogenase and formed by the self‐assembly of 60 subunits, has been functionalized with non‐native iron‐mineralization capability by incorporating two types of iron‐binding peptides. The non‐native peptides introduced at the interior surface do not affect the self‐assembly of E2 protein subunits. In contrast to the wild‐type, the engineered E2 protein cages can serve as size‐ and shape‐constrained reactors for the synthesis of iron nanoparticles. Electrostatic interactions between anionic amino acids and cationic iron molecules drive the formation of iron oxide nanoparticles within the engineered E2 protein cages. The work expands the investigations on nanomaterial biosynthesis using engineered host‐guest encapsulation properties of protein cages.     

Higher Order Assembly of Virus‐like Particles (VLPs) Mediated by Multi‐valent Protein Linkers

Two‐ and three‐dimensional assembly of nanoparticles has generated significant interest because these higher order structures could exhibit collective behaviors/properties beyond those of the individual nanoparticles. Highly specific interactions between molecules, which biology exploits to regulate molecular assemblies such as DNA hybridization, often provide inspiration for the construction of higher order materials using bottom‐up approaches. In this study, higher order assembly of virus‐like particles (VLPs) derived from the bacteriophage P22 is demonstrated by using a small adaptor protein, Dec, which binds to symmetry specific sites on the P22 capsid. Two types of connector proteins, which have different number of P22 binding sites and different geometries (ditopic linker with liner geometry and tetratopic linker with tetrahedral geometry) have been engineered through either a point mutation of Dec or genetic fusion with another protein, respectively. Bulk assembly and layer‐by‐layer deposition of P22 VLPs from solution was successfully achieved using both of the engineered multi‐topic linker molecules, while Dec with only a single binding site does not mediate P22 assembly. Beyond the two types of linkers developed in this study, a wide range of different connector geometries could be envisioned using a similar engineering approach. This is a powerful strategy to construct higher order assemblies of VLP based nanomaterials.     

A Nanoporous Cytochrome c Film with Highly Ordered Porous Structure for Sensing of Toxic Vapors

Creating well‐ordered nanoporosity in biomolecules promises stability and activity, offering access to an even wider range of application possibilities. Here, the preparation of nanoporous protein films containing cytochrome c protein molecules is reported through a soft‐templating strategy using polystyrene (PS) spheres of different sizes as templates. The stability of the cytochrome c film is demonstrated through electrochemistry studies to show a reusable nature of these films over a long period of time. The size of the PS spheres is varied to tune the pore diameter and the thickness of the cytochrome c films, which are quite stable and highly selective for sensing toxic acidic vapors. The fusion of the templating strategy and the self‐assembly of biomolecules may offer various possibilities by generating a new series of porous biomolecules including enzymes with different molecular weights and diameters, peptides, antibodies, and DNA with interesting catalytic, adsorption, sensing, and electronic properties.     

Tubulin Double Helix: Lateral and Longitudinal Curvature Changes of Tubulin Protofilament

By virtue of their native structures, tubulin dimers are protein building blocks that are naturally preprogrammed to assemble into microtubules (MTs), which are cytoskeletal polymers. Here, polycation‐directed (i.e., electrostatically tunable) assembly of tubulins is demonstrated by conformational changes to the tubulin protofilament in longitudinal and lateral directions, creating tubulin double helices and various tubular architectures. Synchrotron small‐angle X‐ray scattering and transmission electron microscopy reveal a remarkable range of nanoscale assembly structures: single‐ and double‐layered double‐helix tubulin tubules. The phase transitions from MTs to the new assemblies are dependent on the size and concentration of polycations. Two characteristic scales that determine the number of observed phases are the size of polycation compared to the size of tubulin (≈4 nm) and to MT diameter (≈25 nm). This work suggests the feasibility of using polycations that have scissor‐ and glue‐like properties to achieve “programmable breakdown” of protein nanotubes, tearing MTs into double‐stranded tubulins and building up previously undiscovered nanostructures. Importantly, a new role of tubulins is defined as 2D shape‐controllable building blocks for supramolecular architectures. These findings provide insight into the design of protein‐based functional materials, for example, as metallization templates for nanoscale electronic devices, molecular screws, and drug delivery vehicles.     

Mastering Dendrimer Self‐Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing

Self‐assembly is a fundamental concept and a powerful approach in molecular science. However, creating functional materials with the desired properties through self‐assembly remains challenging. In this work, through a combination of experimental and computational approaches, the self‐assembly of small amphiphilic dendrons into nanosized supramolecular dendrimer micelles with a degree of structural definition similar to traditional covalent high‐generation dendrimers is reported. It is demonstrated that, with the optimal balance of hydrophobicity and hydrophilicity, one of the self‐assembled nanomicellar systems, totally devoid of toxic side effects, is able to deliver small interfering RNA and achieve effective gene silencing both in cells – including the highly refractory human hematopoietic CD34⁺ stem cells – and in vivo, thus paving the way for future biomedical implementation. This work presents a case study of the concept of generating functional supramolecular dendrimers via self‐assembly. The ability of carefully designed and gauged building blocks to assemble into supramolecular structures opens new perspectives on the design of self‐assembling nanosystems for complex and functional applications.     

Multi‐Component Organic Layers on Metal Substrates

Increasingly high hopes are being placed on organic semiconductors for a variety of applications. Progress along these lines, however, requires the design and growth of increasingly complex systems with well‐defined structural and electronic properties. These issues have been studied and reviewed extensively in single‐component layers, but the focus is gradually shifting towards more complex and functional multi‐component assemblies such as donor–acceptor networks. These blends show different properties from those of the corresponding single‐component layers, and the understanding on how these properties depend on the different supramolecular environment of multi‐component assemblies is crucial for the advancement of organic devices. Here, our understanding of two‐dimensional multi‐component layers on solid substrates is reviewed. Regarding the structure, the driving forces behind the self‐assembly of these systems are described. Regarding the electronic properties, recent insights into how these are affected as the molecule's supramolecular environment changes are explained. Key information for the design and controlled growth of complex, functional multicomponent structures by self‐assembly is summarized.     

Surface‐Layer Lattices as Patterning Element for Multimeric Extremozymes

A promising new approach for the production of biocatalysts comprises the use of surface‐layer (S‐layer) lattices that present functional multimeric enzymes on their surface, thereby guaranteeing most accurate spatial distribution and orientation, as well as maximal effectiveness and stability of these enzymes. For proof of concept, a tetrameric and a trimeric extremozyme are chosen for the construction of S‐layer/extremozyme fusion proteins. By using a flexible peptide linker, either one monomer of the tetrameric xylose isomerase XylA from the thermophilic Thermoanaerobacterium strain JW/SL‐YS 489 or, in another approach, one monomer of the trimeric carbonic anhydrase from the methanogenic archaeon Methanosarcina thermophila are genetically linked to one monomer of the S‐layer protein SbpA of Lysinibacillus sphaericus CCM 2177. After isolation and purification, the self‐assembly properties of both S‐layer fusion proteins as well as the specific activity of the fused enzymes are confirmed, thus indicating that the S‐layer protein moiety does not influence the nature of the multimeric enzymes and vice versa. By recrystallization of the S‐layer/extremozyme fusion proteins on solid supports, the active enzyme multimers are exposed on the surface of the square S‐layer lattice with 13.1 nm spacing.     

Rational Design of Nanocarriers for Intracellular Protein Delivery

Protein/antibody therapeutics have exhibited the advantages of high specificity and activity even at an extremely low concentration compared to small molecule drugs. However, they are accompanied by unfavorable physicochemical properties such as fragile tertiary structure, large molecular size, and poor penetration of the membrane, and thus the clinical use of protein drugs is hindered by inefficient delivery of proteins into the host cells. To overcome the challenges associated with protein therapeutics and enhance their biopharmaceutical applications, various protein‐loaded nanocarriers with desired functions, such as lipid nanocapsules, polymeric nanoparticles, inorganic nanoparticles, and peptides, are developed. In this review, the different strategies for intracellular delivery of proteins are comprehensively summarized. Their designed routes, mechanisms of action, and potential therapeutics in live cells or in vivo are discussed in detail. Furthermore, the perspective on the new generation of delivery systems toward the emerging area of protein‐based therapeutics is presented as well.     

Molecular Self‐Assembly on Graphene

The formation of ordered arrays of molecules via self‐assembly is a rapid, scalable route towards the realization of nanoscale architectures with tailored properties. In recent years, graphene has emerged as an appealing substrate for molecular self‐assembly in two dimensions. Here, the first five years of progress in supramolecular organization on graphene are reviewed. The self‐assembly process can vary depending on the type of graphene employed: epitaxial graphene, grown in situ on a metal surface, and non‐epitaxial graphene, transferred onto an arbitrary substrate, can have different effects on the final structure. On epitaxial graphene, the process is sensitive to the interaction between the graphene and the substrate on which it is grown. In the case of graphene that strongly interacts with its substrate, such as graphene/Ru(0001), the inhomogeneous adsorption landscape of the graphene moiré superlattice provides a unique opportunity for guiding molecular organization, since molecules experience spatially constrained diffusion and adsorption. On weaker‐interacting epitaxial graphene films, and on non‐epitaxial graphene transferred onto a host substrate, self‐assembly leads to films similar to those obtained on graphite surfaces. The efficacy of a graphene layer for facilitating planar adsorption of aromatic molecules has been repeatedly demonstrated, indicating that it can be used to direct molecular adsorption, and therefore carrier transport, in a certain orientation, and suggesting that the use of transferred graphene may allow for predictible molecular self‐assembly on a wide range of surfaces.     

Capillary Force‐Driven,Hierarchical Co‐Assembly of Dandelion‐Like Peptide Microstructures

The wetting and drying of drops on flexible fibers occurs ubiquitously in nature, and the capillary force underlying this phenomenon has motivated our great interest in learning how to direct supramolecular self‐assembly. Here, the hierarchical co‐assembly of two aromatic peptides, diphenylalanine (FF) and ferrocene‐diphenylalanine (Fc‐FF), is reported via sequential, combinatorial assembly. The resulting dandelion‐like microstructures have highly complex architectures, where FF microtube arrays serve as the scapes and the Fc‐FF nanofibers serve as the flower heads. Homogeneous FF microtubes with diameters tailored between 1 and 9 μm and wall thickness ranging from 70 to 950 nm are initially formed by controlling the degree of supersaturation of the FF and the water content. Once the FF microtubes are formed, the growth of the dandelion‐like microstructures is then driven by the capillary force, derived from the wetting and drying of the Fc‐FF solution on the FF microtubes. This simple and ingenious strategy offers many opportunities to develop new and creative methods for controlling the hierarchical self‐assembly of peptides and thus building highly complex nano and microstructures.     

pH‐Regulated Heterostructure Porous Particles Enable Similarly Sized Protein Separation

Porous particles are frequently used for various healthcare applications that involve protein separation processes. However, conventional porous particles, either homogeneous particles or those subjected to surface modification with a layer of specific molecules, often encounter bottlenecks in separating proteins with similar size. Here, it is reported that heterostructure‐enabled separation particles (HESP), synthesized by a double emulsion interfacial polymerization process, can effectively and rapidly separate similarly sized proteins. Double emulsion interfacial polymerization endows the HESP with a nanoscale carboxylic layer outside the particles and inside the pores, allowing pH‐regulated selective adsorption of proteins. Thus, by optimizing the environmental pH, proteins with similar size can be effectively and rapidly separated. These HESP are expected to show potential in widespread applications ranging from biomolecule adsorption, encapsulation, and separation to controlled release and other biomedical fields.     

Tailored Porphyrin Assembly at the Oil–Aqueous Interface Based on the Receding of Three‐Phase Contact Line of Droplet Template

A novel and robust approach to controllable porphyrin assembly is developed at the oil–aqueous interface by tuning the receding of three‐phase contact line of droplet template. The assembled morphologies (ring–, wheel–, and hole–shell shape), aggregate modes and crystal states of porphyrin molecules can be effectively tailored by modulating the receding behavior of three‐phase contact line, which can be realized by varying the hysteresis behavior of substrate or solvent ratios. The developed method for tailored porphyrin assembly will offer an important insight for the design and creation of novel structures.     

Efficient Aggregation‐Induced Emission Manipulated by Polymer Host Materials

Linear copolymer hosts bearing a number of pillar[5]arene dangling side chains are synthesized for the facile construction of highly emissive supramolecular polymer networks (SPNs) upon noncovalently cross‐linking with a series of tetraphenyethylene (TPE)‐based tetratopic guests terminated with different functional groups through supramolecular host–guest interactions. An extremely high fluorescence quantum yield (98.22%) of the SPNs materials is obtained in tetrahydrofuran (THF) by fine‐tuning the parameters, and meanwhile supramolecular light‐harvesting systems based on spherical supramolecular nanoparticles are constructed by interweaving 9,10‐distyrylanthracene (DSA) and TPE‐based guest molecules of aggregation‐induced emission (AIE) with the copolymer hosts in the mixed solvent of THF/H₂O. The present study not only illustrates the restriction of the intramolecular rotations (RIR)‐ruled emission enhancement mechanism regulated particularly by macrocyclic arene‐containing copolymer hosts, but also suggests a new self‐assembly approach to construct high‐performance light‐harvesting materials.     

Self‐Assembly of Shaped Nanoparticles into Free‐Standing 2D and 3D Superlattices

This article describes a novel supramolecular assembly‐mediated strategy for the organization of Au nanoparticles (NPs) with different shapes (e.g., spheres, rods, and cubes) into large‐area, free‐standing 2D and 3D superlattices. This robust approach involves two major steps: (i) the organization of polymer‐tethered NPs within the assemblies of supramolecular comblike block copolymers (CBCPs), and (ii) the disassembly of the assembled CBCP structures to produce free‐standing NP superlattices. It is demonstrated that the crystal structures and lattice constants of the superlattices can be readily tailored by varying the molecular weight of tethered polymers, the volume fraction of NPs, and the matrix of CBCPs. This template‐free approach may open a new avenue for the assembly of NPs into 2D and 3D structures with a wide range of potential applications.     

On the computation of the coupled thermo‐electromagnetic response of continua with particulate microstructure

Recently, several applications, primarily driven by micro‐technology, have emerged where the use of materials with tailored electromagnetic properties is necessary for a successful design. The ‘tailored’ properties are achieved by doping an easily moldable base matrix with particles having dielectric constants that are chosen to give overall desired properties. In many cases, the analysis of such materials requires the simulation of the macroscopic and microscopic electromagnetic response, as well as its resulting coupled thermal response, which can be important to determine possible failure in ‘hot spots’. In this study, a model and a solution strategy are developed to compute the response of a class of fully coupled electro‐magneto‐thermal systems composed of heterogeneous materials, involving the absorption of electromagnetic energy, its conversion to heat and changes in the electromagnetic material properties. The algorithm involves recursive staggering, whose convergence is dependent on the discretized time‐step size. The multifield system coupling can change, becoming weaker, stronger or alternating back and forth. Therefore, it is quite difficult to determine a priori the time‐step size needed to meet a prespecified tolerance on the staggering error, i.e. the incomplete resolution of the coupling between the fields. The presented solution process involves time‐step size adaptivity to control the contraction mapping constant of the multifield system operator in order to induce desired staggering rates of convergence within each time step and to control the staggering error. Three‐dimensional numerical experiments are performed to illustrate the behavior of the model and the solution strategy. Copyright © 2008 John Wiley & Sons, Ltd.     

Solid‐Supported Biomolecules on Modified Silica Surfaces—A Tool for Fast Physicochemical Characterization and High‐Throughput Screening

Biofunctionalization for a wide variety of applications can be achieved by coating silica surfaces with biomolecules such as lipids or proteins. However, specific surface optimization of the inorganic SiO₂ is necessary to achieve biocompatible surfaces. Surface shielded porous silica beads can be non‐covalently coated with a single lipid bilayer. The lipids retain their fluidity in this handy solid‐supported system, perfectly mimicking the soft‐surface properties of cellular membranes. A supramolecular architecture can also be used for functional immobilization of membrane proteins: An artificial cytosolic compartment can be created with the aid of polymers; coating by lipid membranes and integration of membrane proteins results in a solid‐supported biofunctional cellular surface. Another surface modification enables a direct immobilization of human serum albumin (HSA) molecules onto silica surfaces. The HSA on this otherwise passivated surface provides a convenient material for the investigation of unspecific protein binding of pharmaceuticals on a high‐throughput scale.     

Engineering Protein Nanoparticles Out from Components of the Human Microbiome

Nanoscale protein materials are highly convenient as vehicles for targeted drug delivery because of their structural and functional versatility. Selective binding to specific cell surface receptors and penetration into target cells require the use of targeting peptides. Such homing stretches should be incorporated to larger proteins that do not interact with body components, to prevent undesired drug release into nontarget organs. Because of their low interactivity with human body components and their tolerated immunogenicity, proteins derived from the human microbiome are appealing and fully biocompatible building blocks for the biofabrication of nonreactive, inert protein materials within the nanoscale. Several phage and phage‐like bacterial proteins with natural structural roles are produced in Escherichia coli as polyhistidine‐tagged recombinant proteins, looking for their organization as discrete, nanoscale particulate materials. While all of them self‐assemble in a variety of sizes, the stability of the resulting constructs at 37 °C is found to be severely compromised. However, the fine adjustment of temperature and Zn²⁺ concentration allows the formation of robust nanomaterials, fully stable in complex media and under physiological conditions. Then, microbiome‐derived proteins show promise for the regulatable construction of scaffold protein nanomaterials, which can be tailored and strengthened by simple physicochemical approaches.     

Under Diffusion Control: from Structuring Matter to Directional Motion

Self‐organization in synthetic chemical systems is quickly developing into a powerful strategy for designing new functional materials. As self‐organization requires the system to exist far from thermodynamic equilibrium, chemists have begun to go beyond the classical equilibrium self‐assembly that is often applied in bottom‐up supramolecular synthesis, and to learn about the surprising and unpredicted emergent properties of chemical systems that are characterized by a higher level of complexity and extended reactivity networks. The present review focuses on self‐organization in reaction‐diffusion systems. Selected examples show how the emergence of complex morphogenesis is feasible in synthetic systems leading to hierarchically and nanostructured matter. Starting from well‐investigated oscillating reactions, recent developments extend diffusion‐limited reactivity to supramolecular systems. The concept of dynamic instability is introduced and illustrated as an additional tool for the design of smart materials and actuators, with emphasis on the realization of motion even at the macroscopic scale. The formation of spatio‐temporal patterns along diffusive chemical gradients is exploited as the main channel to realize symmetry breaking and therefore anisotropic and directional mechanical transformations. Finally, the interaction between external perturbations and chemical gradients is explored to give mechanistic insights in the design of materials responsive to external stimuli.