Self‐Assembly Toolbox of Tailored Supramolecular Architectures Based on an Amphiphilic Protein Library

The molecular structuring of complex architectures and the enclosure of space are essential requirements for technical and living systems. Self‐assembly of supramolecular structures with desired shape, size, and stability remains challenging since it requires precise regulation of physicochemical and conformational properties of the components. Here a general platform for controlled self‐assembly of tailored amphiphilic elastin‐like proteins into desired supramolecular protein assemblies ranging from spherical coacervates over molecularly defined twisted fibers to stable unilamellar vesicles is introduced. The described assembly protocols efficiently yield protein membrane–based compartments (PMBC) with adjustable size, stability, and net surface charge. PMBCs demonstrate membrane fusion and phase separation behavior and are able to encapsulate structurally and chemically diverse cargo molecules ranging from small molecules to naturally folded proteins. The ability to engineer tailored supramolecular architectures with defined fusion behavior, tunable properties, and encapsulated cargo paves the road for novel drug delivery systems, the design of artificial cells, and confined catalytic nanofactories.     

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.     

Influence of the Thermodynamic and Kinetic Control of Self‐Assembly on the Microstructure Evolution of Silk‐Elastin‐Like Recombinamer Hydrogels

Complex recombinant biomaterials that merge the self‐assembling properties of different (poly)peptides provide a powerful tool for the achievement of specific structures, such as hydrogel networks, by tuning the thermodynamics and kinetics of the system through a tailored molecular design. In this work, elastin‐like (EL) and silk‐like (SL) polypeptides are combined to obtain a silk‐elastin‐like recombinamer (SELR) with dual self‐assembly. First, EL domains force the molecule to undergo a phase transition above a precise temperature, which is driven by entropy and occurs very fast. Then, SL motifs interact through the slow formation of β‐sheets, stabilized by H‐bonds, creating an energy barrier that opposes phase separation. Both events lead to the development of a dynamic microstructure that evolves over time (until a pore size of 49.9 ± 12.7 µm) and to a delayed hydrogel formation (obtained after 2.6 h). Eventually, the network is arrested due to an increase in β‐sheet secondary structures (up to 71.8 ± 0.8%) within SL motifs. This gives a high bond strength that prevents the complete segregation of the SELR from water, which results in a fixed metastable microarchitecture. These porous hydrogels are preliminarily tested as biomimetic niches for the isolation of cells in 3D cultures.     

A Generic Self‐Assembly Process in Microcompartments and Synthetic Protein Nanotubes

Bacterial microcompartments enclose a biochemical pathway and reactive intermediate within a protein envelope formed by the shell proteins. Herein, the orientation of the propanediol‐utilization (Pdu) microcompartment shell protein PduA in bacterial microcompartments and in synthetic nanotubes, and the orientation of PduB in synthetic nanotubes are revealed. When produced individually, PduA hexamers and PduB trimers, tessellate to form flat sheets in the crystal, or they can self‐assemble to form synthetic protein nanotubes in solution. Modelling the orientation of PduA in the 20 nm nanotube so as to preserve the shape complementarity and key interactions seen in the crystal structure suggests that the concave surface of the PduA hexamer faces out. This orientation is confirmed experimentally in synthetic nanotubes and in the bacterial microcompartment produced in vivo. The PduB nanotubes described here have a larger diameter, 63 nm, with the concave surface of the trimer again facing out. The conserved concave surface out characteristic of these nano‐structures reveals a generic assembly process that causes the interface between adjacent subunits to bend in a common direction that optimizes shape complementarity and minimizes steric clashes. This understanding underpins engineering strategies for the biotechnological application of protein nanotubes.     

Self‐Assembling Proteins as High‐Performance Substrates for Embryonic Stem Cell Self‐Renewal

The development of extracellular matrix mimetics that imitate niche stem cell microenvironments and support cell growth for technological applications is intensely pursued. Specifically, mimetics are sought that can enact control over the self‐renewal and directed differentiation of human pluripotent stem cells (hPSCs) for clinical use. Despite considerable progress in the field, a major impediment to the clinical translation of hPSCs is the difficulty and high cost of large‐scale cell production under xeno‐free culture conditions using current matrices. Here, a bioactive, recombinant, protein‐based polymer, termed ZT^Fn, is presented that closely mimics human plasma fibronectin and serves as an economical, xeno‐free, biodegradable, and functionally adaptable cell substrate. The ZT^Fn substrate supports with high performance the propagation and long‐term self‐renewal of human embryonic stem cells while preserving their pluripotency. The ZT^Fn polymer can, therefore, be proposed as an efficient and affordable replacement for fibronectin in clinical grade cell culturing. Further, it can be postulated that the ZT polymer has significant engineering potential for further orthogonal functionalization in complex cell applications.     

Independent Blue and Red Light Triggered Narcissistic Self‐Sorting Self‐Assembly of Colloidal Particles

The ability of living systems to self‐sort different cells into separate assemblies and the ability to independently regulate different structures are one ingredient that gives rise to their spatiotemporal complexity. Here, this self‐sorting behavior is replicated in a synthetic system with two types of colloidal particles; where each particle type independently self‐assembles either under blue or red light into distinct clusters, known as narcissistic self‐sorting. For this purpose, each particle type is functionalized either with the light‐switchable protein VVDHigh or Cph1, which homodimerize under blue and red light, respectively. The response to different wavelengths of light and the high specificity of the protein interactions allows for the independent self‐assembly of each particle type with blue or red light and narcissistic self‐sorting. Moreover, as both of the photoswitchable protein interactions are reversible in the dark; also, the self‐sorting is reversible and dynamic. Overall, the independent blue and red light controlled self‐sorting in a synthetic system opens new possibilities to assemble adaptable, smart, and advanced materials similar to the complexity observed in tissues.     

A S‐Layer Protein of Bacillus anthracis as a Building Block for Functional Protein Arrays by In Vitro Self‐Assembly

S‐layer proteins create a cell‐surface layer architecture in both bacteria and archaea. Because S‐layer proteins self‐assemble into a native‐like S‐layer crystalline structure in vitro, they are attractive building blocks in nanotechnology. Here, the potential use of the S‐layer protein EA1 from Bacillus anthracis in constructing a functional nanostructure is investigated, and apply this nanostructure in a proof‐of‐principle study for serological diagnosis of anthrax. EA1 is genetically fused with methyl parathion hydrolase (MPH), to degrade methyl parathion and provide a label for signal amplification. EA1 not only serves as a nanocarrier, but also as a specific antigen to capture anthrax‐specific antibodies. As results, purified EA1–MPH forms a single layer of crystalline nanostructure through self‐assembly. Our chimeric nanocatalyst greatly improves enzymatic stability of MPH. When applied to the detection of anthrax‐specific antibodies in serum samples, the detection of our EA1–MPH nanostructure is nearly 300 times more sensitive than that of the unassembled complex. Together, it is shown that it is possible to build a functional and highly sensitive nanosensor based on S‐layer protein. In conclusion, our present study should serve as a model for the development of other multifunctional nanomaterials using S‐layer proteins.