Jianpeng Wang

A general strategy for site-directed enzyme immobilization by using NiO nanoparticle decorated mesoporous silica.

Mesoporous materials have recently gained much attention owing to their large surface area, narrow pore size distribution, and superior pore structure. These materials have been demonstrated as excellent solid supports for immobilization of a variety of proteins and enzymes for their potential applications as biocatalysts in the chemical and pharmaceutical industries. However, the lack of efficient and reproducible methods for immobilization has limited the activity and recyclability of these biocatalysts. Furthermore, the biocatalysts are usually not robust owing to their rapid denaturation in bulk solvents. To solve these problems, we designed a novel hybrid material system, mesoporous silica immobilized with NiO nanoparticles (SBA-NiO), wherein enzyme immobilization is directed to specific sites on the pore surface of the material. This yielded the biocatalytic species with higher activity than free enzyme in solution. These biocatalytic species are recyclable with minimal loss of activity after several cycles, demonstrating an advantage over free enzymes.

Cascade Biocatalysis by Multienzyme–Nanoparticle Assemblies

Multienzyme complexes are of paramount importance in biosynthesis in cells. Yet, how sequential enzymes of cascade catalytic reactions synergize their activities through spatial organization remains elusive. Recent development of site-specific protein-nanoparticle conjugation techniques enables us to construct multienzyme assemblies using nanoparticles as the template. Sequential enzymes in menaquinone biosynthetic pathway were conjugated to CdSe-ZnS quantum dots (QDs, a nanosized particulate material) through metal-affinity driven self-assembly. The assemblies were characterized by electrophoretic methods, the catalytic activities were monitored by reverse-phase chromatography, and the composition of the multienzyme-QD assemblies was optimized through a progressive approach to achieve highly efficient catalytic conversion. Shorter enzyme-enzyme distance was discovered to facilitate intermediate transfer, and a fine control on the stoichiometric ratio of the assembly was found to be critical for the maximal synergy between the enzymes. Multienzyme-QD assemblies thereby provide an effective model to scrutinize the synergy of cascade enzymes in multienzyme complexes.

Assembly of Multivalent Protein Ligands and Quantum Dots: A Multifaceted Investigation

The development of multivalent protein ligands for nanoparticles lags behind that of multidentate polymers and small-molecule ligands largely because of a lack of thorough understanding of the interaction between nanoparticles and multimeric proteins. Guided by protein crystal structures, we have harnessed recombinant technology to develop a collection of mCherry fused multimeric proteins with different spatial distributions of the quantum dot (QD)-binding sequence, hexahistidine tag (histag). All of the proteins can behave as ligands to assemble with ZnS-CdSe QDs through metal-affinity-driven self-assembly. We have observed that protein shape and geometry greatly affect the stoichiometry and stability of their assemblies with QDs. We also demonstrate a peptide-induced structural transition of a nanobelt protein that preorganizes the QD-binding sites and effects a more efficient assembly with QDs. This work reports the first multifaceted investigation on how multivalent proteins, in particular, dimers, tetramers, and linear multidentate proteins, assemble with QDs. It also manifests our capability of harnessing structural and conformational information about proteins to design multivalent protein ligands for QD surface functionalization.

Affinity-Guided Covalent Conjugation Reactions Based on PDZ–Peptide and SH3–Peptide Interactions

Specific protein-peptide interactions are prevalent in the living cells and form a tightly regulated signaling network. These interactions, many of which have structural information revealed, provide ideal templates for affinity-guided covalent bioconjugation. Here we report the development of a set of four new reactions that covalently and site-specifically link nonenzymatic scaffolding domains (two PDZ and two SH3 domains) and their ligands through thiol-chloroacetyl SN2 reaction. Guided by the three-dimensional structure of the wild type complex, a selected position of the protein was mutated to cysteine, and at the same time, an α-chloroacetyl group was installed at a corresponding position of the peptide. Specific binding interaction between the two brings the reactive groups into close proximity, converts the nonreactive cysteine residue into a content-dependent reactive site, and induces the nucleophilic reaction that is inert in the absence of the binding event. The specificity, orthogonality, and modularity of the four reactions were characterized, the reaction was applied to label proteins in vitro and receptor on the surface of mammalian cells, and the system was utilized to assemble covalent protein complexes with unnatural geometries.

Simultaneous monitoring of quantum dots and their assembly and disassembly with PreScission protease using capillary electrophoresis with fluorescence detection

A novel assay was developed for the simultaneous monitoring of quantum dots and their assembly and disassembly with PreScission protease using capillary electrophoresis with fluorescence detection. Quantum dots and PreScission protease were injected into a capillary sequentially, then mixed and assembled via a thioether bond upon coupling to glutathione S-transferase tag inside the capillary. The in-capillary assembly was influenced by the molar ratio and the time interval of injection. Furthermore, the simultaneous monitoring of quantum dots and their assembly with PreScission protease and glutathione induced disassembly was achieved by adjusting the sampling sequence and the time interval of injection. More importantly, the in-capillary assay could be also applied to the online detection of glutathione.

In-capillary probing of quantum dots and fluorescent protein self-assembly and displacement using Förster resonance energy transfer.

Herein, a Förster resonance energy transfer system was designed, which consisted of CdSe/ZnS quantum dots donor and mCherry fluorescent protein acceptor. The quantum dots and the mCherry proteins were conjugated to permit Förster resonance energy transfer. Capillary electrophoresis with fluorescence detection was used for the analyses for the described system. The quantum dots and mCherry were sequentially injected into the capillary, while the real-time fluorescence signal of donor and acceptor was simultaneously monitored by two channels with fixed wavelength detectors. An effective separation of complexes from free donor and acceptor was achieved. Results showed quantum dots and hexahistidine tagged mCherry had high affinity and the assembly was affected by His6 -mCherry/quantum dot molar ratio. The kinetics of the self-assembly was calculated using the Hill equation. The microscopic dissociation constant values for out of- and in-capillary assays were 10.49 and 23.39 μM, respectively. The capillary electrophoresis with fluorescence detection that monitored ligands competition assay further delineated the different binding capacities of histidine containing peptide ligands for binding sites on quantum dots. This work demonstrated a novel approach for the improvement of Förster resonance energy transfer for higher efficiency, increased sensitivity, intuitionistic observation, and low sample requirements of the in-capillary probing system.

Investigation of multivalent interactions between conjugate of quantum dots with c-Myc peptide tag and the anti-c-Myc antibody by capillary electrophoresis with fluorescence detection

Herein, we report an assay for detecting the binding of a multivalent peptide and antibody within a capillary with the use of fluorescence coupled capillary electrophoresis. Quantum dots and a c-Myc tag containing peptide EQKLISEEDLG4 H6 were injected sequentially and formed a multivalent quantum dot-EQKLISEEDLG4 H6 assembly within the capillary. The efficiency of the quantum dot-peptide self-assembly was affected by the peptide/quantum dot molar ratio, sampling time, and interval time. Finally, the binding of the monoclonal anti-c-Myc antibody and the multivalent quantum dot-EQKLISEEDLG4 H6 ligand was studied using an in-capillary assay. The microscopic dissociation constant for the self-assembly of monoclonal anti-c-Myc antibody and quantum dot-EQKLISEEDLG4 H6 was determined to be 14.1 μM with a stoichiometry of the peptide-antibody complex of 1.7 determined after fitting this to the Hill equation. This method can be further extended to detect a wide range of biomolecule-biomolecule binding interactions.

Online probing quantum dots and engineered enzyme self‐assembly in a nanoliter scale

Nanoparticles provide significantly enhanced binding characteristics. However, fast online probing of the self‐assembly process remains hard to achieve in practice. Herein, we report a fluorescence coupled CE method for probing the self‐assembly events between quantum dots (QDs) and engineered Jumonji domain‐containing protein 6 (Jmjd6) enzyme. QDs and Jmjd6 were sequentially injected into the capillary, where the self‐assembly took place in a nanoliter scale. In particular, we showed that the Jmjd6/QD ratio, the interval time, and the injection volume had a great effect on the online self‐assembly. The current approach may allow for a better understanding of QDs–enzyme self‐assembly and enzymatic activity detection.

Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G-quadruplex aptamer and thrombin

The dynamic binding status between the thrombin and its G-quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence-coupled capillary electrophoresis method. A 29-nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G-quadruplex from unstructured 29-nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6-carboxyfluorescein labeled G-quadruplex aptamer was measured. Third, the stability of G-quadruplex aptamer-thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29-nucleic acid thrombin binding aptamer could compete with G-quadruplex aptamer and thus disassociated the G-quadruplex structure into an unstructured aptamer. These data suggest that our in-house established fluorescence-coupled capillary electrophoresis assay could be applied to binding studies of the G-quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available.

Investigation of the weak binding of a tetrahistidine‐tagged peptide to quantum dots by using capillary electrophoresis with fluorescence detection

Capillary electrophoresis with fluorescence detection was utilized to probe the self-assembly between cyanine group dye labeled tetrahistidine containing peptide and CdSe/ZnS quantum dots, inside the capillary. Quantum dots and cyanine group dye labeled tetrahistidine containing peptide were injected into the capillary one after the other and allowed to self-assemble. Their self-assembly resulted into a measurable Förster resonance energy transfer signal between quantum dots and cyanine group dye labeled tetrahistidine containing peptide. The Förster resonance energy transfer signal increased upon increasing the cyanine group dye labeled tetrahistidine containing peptide/quantum dot molar ratio and reached a plateau at the 32/1 molar ratio. Additionally, the Förster resonance energy transfer signal was also affected by the increment of the interval time of injection and the sampling time. Online ligand exchange experiments were used to assess, the potential of a monovalent ligand of imidazole and a hexavalent ligand peptide, to displace surface bound cyanine group dye labeled peptide ligands from the quantum dots surface. Under optimal conditions, a linear relationship between the integrated peak areas and hexavalent ligand peptide was obtained at a hexavalent ligand concentration range of 0-0.5 mM. Therefore, the present assay has the potential to be applied in the online ligands detection.