The phase behaviour of mCherry-b-PNIPAM (mChP) block copolymers with four different PNIPAM coil fractions is investigated in concentrated aqueous solution as a function of both concentration and temperature, demonstrating both order–order transitions (OOTs) and order–disorder transitions (ODTs) in globular protein–polymer block copolymers. Independent of coil volume fraction from 0.25 to 0.70, the temperature–concentration phase diagrams share several common features. At low concentrations, mCherry-b-PNIPAM forms a homogeneous disordered phase, and macrophase separation into an ordered conjugate-rich phase and a solvent-rich phase is observed at temperatures above the PNIPAM thermoresponsive transition temperature. mChP solutions are also observed to undergo a low-temperature ODT driven by increasing concentration. The order–disorder transition concentration (ODTC) behaviour of mChP is minimized for symmetric conjugates, suggesting that repulsive solvent-mediated protein–polymer interactions provide a driving force for self-assembly. Both coil fraction and solvent selectivity have large effects on the morphologies formed—disordered micelles, hexagonally packed cylinders, lamellae, and perforated lamellae are identified with the combination of small-angle X-ray scattering (SAXS), depolarized light scattering (DPLS), turbidimetry, and differential scanning calorimetry (DSC). An OOT is observed upon increasing temperature for three of the studied coil fractions at concentrations of 40–50 wt% due to changing solvent selectivity. SANS contrast-matching experiments show that water is weakly selective for PNIPAM at low temperatures and strongly selective for mCherry at high temperatures.

/pdf/phase-transitions-in-concentrated-solution-self-assembly-of-431jbq485j.pdf

Phase Transitions in Concentrated Solution Self-Assembly of Globular Protein-Polymer Block Copolymers

This review presents precisely defined amphiphilic dendrons, their self-association properties, and their different uses. Dendrons, also named dendritic wedges, are composed of a core having two different types of functions, of which one type is used for growing or grafting branched arms, generally multiplied by 2 at each layer by using 1→2 branching motifs. A large diversity of structures has been already synthesized. In practically all cases, their synthesis is based on the synthesis of known dendrimers, such as poly(aryl ether), poly(amidoamine) (in particular PAMAM), poly(amide) (in particular poly(L-lysine)), 1→3 branching motifs (instead of 1→2), poly(alkyl ether) (poly(glycerol) and poly(ethylene glycol)), poly(ester), and those containing main group elements (poly(carbosilane) and poly(phosphorhydrazone)). In most cases, the hydrophilic functions are on the surface of the dendrons, whereas one or two hydrophobic tails are linked to the core. Depending on the structure of the dendrons, and on the experimental conditions used, the amphiphilic dendrons can self-associate at the air-water interface, or form micelles (eventually tubular, but most generally spherical), or form vesicles. These associated dendrons are suitable for the encapsulation of low-molecular or macromolecular bioactive entities to be delivered in cells. This review is organized depending on the nature of the internal structure of the amphiphilic dendrons (aryl ether, amidoamine, amide, quaternary carbon atom, alkyl ether, ester, main group element). The properties issued from their self-associations are described all along the review.

Supramolecular Self-Associations of Amphiphilic Dendrons and Their Properties.

Supramolecular assembly of biomolecules/macromolecules stems from the desire to mimic complex biological structures and functions of living organisms. While DNA nanotechnology is already in an advanced stage, protein assembly is still in its infancy as it is a significantly difficult task due to their large molecular weight, conformational complexity and structural instability towards variation in temperature, pH or ionic strength. This article reports highly stable redox-responsive supramolecular assembly of a protein Bovine serum albumin (BSA) which is functionalized with a supramolecular structure directing unit (SSDU). The SSDU consists of a benzamide functionalized naphthalene-diimide (NDI) chromophore which is attached with the protein by a bio-reducible disulfide linker. The SSDU attached protein (NDI-BSA) exhibits spontaneous supramolecular assembly in water by off-set π-stacking among the NDI chromophores, leading to the formation of spherical nanoparticles (diameter: 150–200 nm). The same SSDU when connected with a small hydrophilic wedge (NDI-1) instead of the large globular protein, exhibits a different π-stacking mode with relatively less longitudinal displacement which results in a fibrillar network and hydrogelation. Supramolecular co-assembly of NDI-BSA and NDI-1 (3 : 7) produces similar π-stacking and an entangled 1D morphology. Both the spherical assembly of NDI-BSA or the fibrillar co-assembly of NDI-BSA + NDI-1 (3 : 7) provide sufficient thermal stability to the protein as its thermal denaturation could be completely surpassed while the secondary structure remained intact. However, the esterase like activity of the protein reduced significantly as a result of such supramolecular assembly indicating limited access by the substrate to the active site of the enzyme located in the confined environment. In the presence of glutathione (GSH), a biologically important tri-peptide, due to the cleavage of the disulfide bond, the protein became free and was released, resulting in fully regaining its enzymatic activity. Such supramolecular assembly provided excellent protection to the protein against enzymatic hydrolysis as the relative hydrolysis was estimated to be <30% for the co-assembled protein with respect to the free protein under identical conditions. Similar to bioactivity, the enzymatic hydrolysis also became prominent after GSH-treatment, confirming that the lack of hydrolysis in the supramolecularly assembled state is indeed related to the confinement of the protein in the nanostructure assembly.

/pdf/redox-responsive-activity-regulation-in-exceptionally-stable-xuzg4m0j39.pdf

Redox responsive activity regulation in exceptionally stable supramolecular assembly and co-assembly of a protein

The rational design of a multi-responsive protein-based supramolecular system that can predictably respond to more than one stimulus remains an essential but highly challenging goal in biomolecular engineering. Herein, we report a novel chemical method for the construction of multi-responsive supramolecular nanoassemblies using custom-designed facially amphiphilic monodisperse protein-dendron bioconjugates. The macromolecular synthons contain a globular hydrophilic protein domain site-specifically conjugated to photo-responsive hydrophobic benzyl-ether dendrons of different generations through oligo(ethylene glycol) linkers of defined length. The size of the protein nanoassemblies can be systematically tuned by choosing an appropriate dendron or linker of defined length. Exposure of protein nanoassemblies to light results in partial rather than complete disassembly of the complex. The newly formed protein nanoparticle no longer responds to light but could be disassembled into constitutive monomers under acidic conditions or by further treatment with a small molecule. More interestingly, the distribution ratio of the assembled versus disassembled states of protein nanoassemblies after photochemical reaction does not depend on dendron generation, the nature of the linker functionality or the identity of the protein, but is heavily influenced by the linker length. In sum, this work discloses a new chemical method for the rational design of a monodisperse multi-responsive protein-based supramolecular system with exquisite control over the disassembly process.

Programmed and Sequential Disassembly of Multi-responsive Supramolecular Protein Nanoassemblies: A Detailed Mechanistic Investigation

The custom design of protein-dendron amphiphilic macromolecules is at the forefront of macromolecular engineering. Macromolecules with this architecture are very interesting because of their ability to self-assemble into various biomimetic nanoscopic structures. However, to date, there are no reports on this concept due to technical challenges associated with the chemical synthesis. Towards that end, herein, a new chemical methodology for the modular synthesis of a suite of monodisperse, facially amphiphilic, protein-dendron bioconjugates is reported. Benzyl ether dendrons of different generations (G1-G4) are coupled to monodisperse cetyl ethylene glycol to form macromolecular amphiphilic activity-based probes (AABPs) with a single protein reactive functionality. Micelle-assisted protein labeling technology is utilized for site-specific conjugation of macromolecular AABPs to globular proteins to make monodisperse, facially amphiphilic, protein-dendron bioconjugates. These biohybrid conjugates have the ability to self-assemble into supramolecular protein nanoassemblies. Self-assembly is primarily mediated by strong hydrophobic interactions of the benzyl ether dendron domain. The size, surface charge, and oligomeric state of protein nanoassemblies could be systematically tuned by choosing an appropriate dendron or protein of interest. This chemical method discloses a new way to custom-make monodisperse, facially amphiphilic, protein-dendron bioconjugates.

Design, Synthesis, and Self‐Assembly Studies of a Suite of Monodisperse, Facially Amphiphilic, Protein–Dendron Conjugates

The self-assembly of proteins into higher-order superstructures is ubiquitous in biological systems. Genetic methods comprising both computational and rational design strategies are emerging as powerful methods for the design of synthetic protein complexes with high accuracy and fidelity. Although useful, most of the reported protein complexes lack a dynamic behavior, which may limit their potential applications. On the contrary, protein engineering by using chemical strategies offers excellent possibilities for the design of protein complexes with stimuli-responsive functions and adaptive behavior. However, designs based on chemical strategies are not accurate and therefore, yield polydisperse samples that are difficult to characterize. Here, we describe simple design principles for the construction of protein complexes through a supramolecular chemical strategy. A micelle-assisted activity-based protein-labeling technology has been developed to synthesize libraries of facially amphiphilic synthetic proteins, which self-assemble to form protein complexes through hydrophobic interaction. The proposed methodology is amenable for the synthesis of protein complex libraries with molecular weights and dimensions comparable to naturally occurring protein cages. The designed protein complexes display a rich structural diversity, oligomeric states, sizes, and surface charges that can be engineered through the macromolecular design. The broad utility of this method is demonstrated by the design of most sophisticated stimuli-responsive systems that can be programmed to assemble/disassemble in a reversible/irreversible fashion by using the pH or light as trigger.

Rational Design of Supramolecular Dynamic Protein Assemblies by Using a Micelle-Assisted Activity-Based Protein-Labeling Technology.

Rational Design of Semi‐Synthetic Protein Complexes with the Defined Oligomeric State

The reversible nature of disulfide functionality has been exploited to design intelligent materials such as nanocapsules, micelles, vesicles, inorganic nanoparticles, peptide and nucleic acid nanodevices. Herein, we report a new chemical methodology for the construction redox-sensitive protein assemblies using monodisperse facially amphiphilic protein-dendron bioconjugates. The disulfide functionality is strategically placed between the dendron and protein domains. The custom designed bioconjugates self-assembled into nanoscopic objects of a defined size dictated by the nature of dendron domain. The stimuli-responsive behavior of the protein assemblies is demonstrated using a suitable redox trigger.

Pavankumar Janardhan Bhandari

Papers

Rational Design of Supramolecular Dynamic Protein Assemblies by Using a Micelle-Assisted Activity-Based Protein-Labeling Technology.

Programmed and Sequential Disassembly of Multi-responsive Supramolecular Protein Nanoassemblies: A Detailed Mechanistic Investigation

Design, Synthesis, and Self‐Assembly Studies of a Suite of Monodisperse, Facially Amphiphilic, Protein–Dendron Conjugates

Rational Design of Semi‐Synthetic Protein Complexes with the Defined Oligomeric State

Rational Design of Programmable Monodisperse Semi-Synthetic Protein Nanomaterials Containing Engineered Disulfide Functionality**