A highly controllable protein self-assembly system with morphological versatility induced by reengineered host-guest interactions.

Manipulating proteins to self-assemble into highly ordered nanostructures not only provides insights into the natural protein assembly process but also allows access to advanced biomaterials. Host-guest interactions have been widely used in the construction of artificial protein assemblies in recent years. CB[8] can selectively associate with two tripeptide Phe-Gly-Gly (FGG) tags with an extraordinarily high binding affinity (Kter = 1.5 × 1011 M-2). However, the FGG tags utilized before are all fixed to the N-termini via genetic fusion; this spatial limitation greatly confined the availability of the CB[8]/FGG pair in the construction of more sophisticated protein nanostructures. Here we first designed and synthesized a maleimide-functionalized Phe-Gly-Gly tag as a versatile site-specific protein modification tool; this designed tag can site-selectively introduce desired guest moieties onto protein surfaces for host-guest driven protein assembly. When regulating the self-assembly process of proteins and CB[8], the constructed protein nanosystem can exhibit distinctive morphological diversities ranging from nanorings, nanospirals, nanowires to superwires. This work developed a new strategy for site-specific protein modification of the CB[8] binding tag and provides a possible direction for the construction of 'smart', dynamic self-assembly systems.

An ion signal responsive dynamic protein nano-spring constructed by high ordered host-guest recognition.

A protein self-assembly nano-spring was developed through host-guest interactions between cucurbit[8]uril and tripeptide FGG tags of fusion protein FGG-recoverin-GST. Fine control of the conformational changes of the Ca(2+)-responsive domain allows for a 50% stretch of the protein nano-spring as it switches from the contracted state to the extended state.

Reversible pH-controlled switching of an artificial antioxidant selenoenzyme based on pseudorotaxane formation and dissociation.

A pH-responsive artificial selenoenzyme was constructed by reversible binding between organoselenium compound a1 nd CB[6] to form a pseudorotaxane-based molecular switch in response to pH stimuli. The glutathione peroxidase (GPx) activity of the artificial selenoenzyme can be switched on/off in a mild and body suitable environment between pH = 7 and pH = 6.

Self-assembly of cricoid proteins induced by "soft nanoparticles": an approach to design multienzyme-cooperative antioxidative systems.

A strategy to construct high-ordered protein nanowires by electrostatic assembly of cricoid proteins and "soft nanoparticles" was developed. Poly(amido amine) (PAMAM) dendrimers on high generation that have been shown to be near-globular macromolecules with all of the amino groups distributing throughout the surface were ideal electropositive "soft nanoparticles" to induce electrostatic assembly of electronegative cricoid proteins. Atomic force microscopy and transmission electron microscopy all showed that one "soft nanoparticle" (generation 5 PAMAM, PD5) could electrostatically interact with two cricoid proteins (stable protein one, SP1) in an opposite orientation to form sandwich structure, further leading to self-assembled protein nanowires. The designed nanostructures could act as versatile scaffolds to develop multienzyme-cooperative antioxidative systems. By means of inducing catalytic selenocysteine and manganese porphyrin to SP1 and PD5, respectively, we successfully designed antioxidative protein nanowires with both excellent glutathione peroxidase and superoxide dismutase activities. Also, the introduction of selenocysteine and manganese porphyrin did not affect the assembly morphologies. Moreover, this multienzyme-cooperative antioxidative system exhibited excellent biological effect and low cell cytotoxicity.

Quantum-dot-induced self-assembly of cricoid protein for light harvesting.

Stable protein one (SP1) has been demonstrated as an appealing building block to design highly ordered architectures, despite the hybrid assembly with other nano-objects still being a challenge. Herein, we developed a strategy to construct high-ordered protein nanostructures by electrostatic self-assembly of cricoid protein nanorings and globular quantum dots (QDs). Using multielectrostatic interactions between 12mer protein nanoring SP1 and oppositely charged CdTe QDs, highly ordered nanowires with sandwich structure were achieved by hybridized self-assembly. QDs with different sizes (QD1, 3-4 nm; QD2, 5-6 nm; QD3, ∼10 nm) would induce the self-assembly protein rings into various nanowires, subsequent bundles, and irregular networks in aqueous solution. Atomic force microscopy, transmission electron microscopy, and dynamic light scattering characterizations confirmed that the size of QDs and the structural topology of the nanoring play critical functions in the formation of the superstructures. Furthermore, an ordered arrangement of QDs provides an ideal scaffold for designing the light-harvesting antenna. Most importantly, when different sized QDs (e.g., QD1 and QD3) self-assembled with SP1, an extremely efficient Förster resonance energy transfer was observed on these protein nanowires. The self-assembled protein nanostructures were demonstrated as a promising scaffold for the development of an artificial light-harvesting system.

Construction of a highly stable artificial glutathione peroxidase on a protein nanoring.

Stable Protein One (SP1) is a boiling-stable oligomeric protein. The unique characteristics of SP1 offer a scaffold to design artificial enzymes against extreme temperature. Here, an efficient antioxidase is successfully constructed on the ring-shaped SP1 homododecamer. By means of computational design and genetic engineering, the active center of glutathione peroxidase (GPx), selenocysteine (Sec), is introduced to the SP1 monomer surface, and the self-assembly properties of the protein monomer lead to a ring-shaped SP1 with homododecamer catalytic selenium centers. This artificial selenoenzyme exhibits high GPx catalytic activity and shows a typical ping-pong kinetic mechanism. Moreover, it has a significantly broader temperature range and high thermostability. Owing to having multi-GPx active centers on a SP1 oligomer, this selenium-containing biomacromolecule exerts an excellent capability to protect cells from oxidative damage at the mitochondrial level. This strategy represents a new way to develop thermostable artificial nanoenzymes for some specific applications.