The key property of protein-nanoparticle conjugates is the bioactivity of the protein. The ability to accurately modulate the activity of protein on the nanoparticles at the interfaces is important in many applications. In the work reported here, modulation of the activity of protein-gold nanoparticle (AuNP) conjugates by specifically orienting the protein and by varying the surface density of the protein was investigated. Different orientations were achieved by introducing cysteine (Cys) residues at specific sites for binding to gold. We chose Escherichia coli inorganic pyrophosphatase (PPase) as a model protein and used site-directed mutagenesis to generate two mutant types (MTs) with a single Cys residue on the surface: MT1 with Cys near the active center and MT2 with Cys far from the active center. The relative activities of AuNP conjugates with wild type (WT), MT1, and MT2 were found to be 44.8%, 68.8%, and 91.2% of native PPase in aqueous solution. Site-directed orientation with the binding site far from the active center thus allowed almost complete preservation of the protein activity. The relative activity of WT and MT2 conjugates did not change with the surface density of the protein, while that of MT1 increased significantly with increasing surface density. These results demonstrate that site-directed orientation and surface density can both modulate the activity of proteins conjugated to AuNP and that orientation has a greater effect than density. Furthermore, increasing the surface density of the specifically oriented protein MT2, while having no significant effect on the specific activity of the protein, still allowed increased protein loading on the AuNP and thus increased the total protein activity. This is of great importance in the study on the interface of protein and nanoparticle and the applications for enzyme immobilization, drug delivery, and biocatalysis.
Block copolymer nanoparticles have been widely used for advanced materials. However, the stabilization is challenging. Herein, we present a method for convenient yet reliable synthesis of stabilized polyion complex (PIC) nanometer-sized spheres and micrometer-sized ultrathin lamellae and vesicles by taking advantage of the wavelength orthogonality of UV-induced disulfide exchange and visible light-initiated polymerization-induced electrostatic self-assembly (PIESA). Disulfide-containing PIC vesicles are synthesized at scale using this PIESA, undergoing a small sphere-to-larger sphere-tolamella-to-vesicle transition. As such, surface-neutralized and surface-charged micrometer-sized vesicles can be achieved. UV irradiation of the vesicles (5.0 mg/mL in water) in ambient air induces very fast exchange reaction of locally confined/enriched disulfide motifs, leading to cross-linking, shape transition, and cystamine salt release in 4 min. As such, cross-linked PIC spheres, lamellae, and vesicles can be achieved, in one pot, from one single vesicle precursor. The wavelength orthogonality is evident from disabled PIESA synthesis under UV light and ineffective postpolymerization functionalization under visible light. The cross-linked PIC spheres and micrometer-sized ultrathin lamellae and vesicles show outstanding shape/size stability and high reversibility in the solution-adaptive electrostatic hierarchical self-assembly and disassembly upon adding ethanol into aqueous dispersion and subsequent dialysis.
It is important to effectively maintain and modulate the bioactivity of protein-nanoparticle conjugates for their further and intensive applications. The strategies of controlling protein activity via "tailor-made surfaces" still have some limitations, such as the difficulties in further modulation of the bioactivity and the proteolysis by some proteases. Thus, it is essential to establish a responsive protein-nanoparticle conjugate system to realize not only controllable modulations of protein activity in the conjugates by incorporating sensitivity to environmental cues but also high resistance to proteases. In the work reported here, Escherichia coli (E. coli) inorganic pyrophosphatase (PPase) and poly(N-isopropylacrylamide) (pNIPAM) were both fabricated onto gold nanoparticles (AuNPs), forming AuNP-PPase-pNIPAM conjugates. The bioactivity-modulating capability of the conjugates with changes in temperature was systematically investigated by varying the molecular weight of pNIPAM, the PPase/pNIPAM molar ratio on AuNP, and the orientation of the proteins. Under proper conditions, the activity of the conjugate at 45 °C was approximately 270% of that at 25 °C. In the presence of trypsin digestion, much less conjugate activity than protein activity was lost. These findings indicate that the fabrication of AuNP-protein-pNIPAM conjugates can both modulate protein activity on a large scale and show much higher resistance to protease digestion, exhibiting great potential in targeted delivery, controllable biocatalysis, and molecular/cellular recognition.
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