Chenghong Lei

Clay Nanoparticle-Supported Single-Molecule Fluorescence Spectroelectrochemistry

Here we report that clay nanoparticles allow formation of a modified transparent electrode, spontaneous adsorption of fluorescent redox molecules on the clay layer, and thus the subsequent observation of single-molecule fluorescence spectroelectrochemistry. We can trace single-molecule fluorescence spectroelectrochemistry by probing the fluorescence intensity change of individually immobilized single redox molecules modulated via cyclic voltammetric potential scanning. This work opens a new approach to explore interfacial electron transfer mechanisms of redox reactions.

The Role of Nonbonded Interactions in the Conformational Dynamics of Organophosphorous Hydrolase Adsorbed onto Functionalized Mesoporous Silica Surfaces

The enzyme organophosphorous hydrolase (OPH) catalyzes the hydrolysis of a wide variety of organophosphorous compounds with high catalytic efficiency and broad substrate specificity. The immobilization of OPH in functionalized mesoporous silica (FMS) surfaces increases significantly its catalytic specific activity, as compared to the enzyme in solution, with important applications for the detection and decontamination of insecticides and chemical warfare agents. Experimental measurements of immobilization efficiency as a function of the charge and coverage percentage of different functional groups have been interpreted as electrostatic forces being the predominant interactions underlying the adsorption of OPH onto FMS surfaces. Explicit solvent molecular dynamics simulations have been performed for OPH in bulk solution and adsorbed onto two distinct interaction potential models of the FMS functional groups to investigate the relative contributions of nonbonded interactions to the conformational dynamics and adsorption of the protein. Our results support the conclusion that electrostatic interactions are responsible for the binding of OPH to the FMS surface. However, these results also show that van der Waals forces are detrimental for interfacial adhesion. In addition, it is found that OPH adsorption onto the FMS models favors a protein conformation whose active site is fully accessible to the substrate, in contrast to the unconfined protein.

Delivery of MicroRNA-10b with Polylysine Nanoparticles for Inhibition of Breast Cancer Cell Wound Healing

Recent studies revealed that micro RNA-10b (mir-10b) is highly expressed in metastatic breast cancer cells and positively regulates breast cancer cell migration and invasion through inhibition of HOXD10 target synthesis. In this study we designed anti-mir-10b molecules and combined them with poly L-lysine (PLL) to test the delivery effectiveness. An RNA molecule sequence exactly matching the mature mir-10b minor antisense showed strong inhibition when mixed with PLL in a wound-healing assay with human breast cell line MDA-MB-231. The resulting PLL-RNA nanoparticles delivered the anti-microRNA molecules into cytoplasm of breast cancer cells in a concentration-dependent manner that displayed sustainable effectiveness.

Probing mechanisms for enzymatic activity enhancement of organophosphorus hydrolase in functionalized mesoporous silica

We have previously reported that organophosphorus hydrolase (OPH) can be spontaneously entrapped in functionalized mesoporous silica (FMS) with HOOC- as the functional groups and the entrapped OPH in HOOC-FMS showed enhanced enzyme specific activity. This work is to study the mechanisms that why OPH entrapped in FMS displayed the enhanced activity in views of OPH-FMS interactions using spectroscopic methods. The circular dichroism (CD) spectra show that, comparing to the secondary structure of OPH free in solution, OPH in HOOC-FMS displayed increased alpha-helix/beta-strand transition of OPH with increased OPH loading density. The fluorescence emission spectra of Trp residues were used to assess the tertiary structural changes of the enzyme. There was a 42% increase in fluorescence. This is in agreement with the fact that the fluorescence intensity of OPH was increased accompanying with the increased OPH activity when decreasing urea concentrations in solution. The steady-state anisotropy was increased after OPH entrapping in HOOC-FMS comparing to the free OPH in solution, indicating that protein mobility was reduced upon entrapment. The solvent accessibility of Trp residues of OPH was probed by using acrylamide as a collisional quencher. Trp residues of OPH-FMS had less solvent exposure comparing with free OPH in solution due to its electrostatical binding to HOOC-FMS thereby displaying the increased fluorescence intensity. These results suggest the interactions of OPH with HOOC-FMS resulted in the protein immobilization and a favorable conformational change for OPH in the crowded confinement space and accordingly the enhanced activity.

Heated Proteins are Still Active in a Functionalized Nanoporous Support

Even under heated conditions, the nearly native conformation and activity of a protein can be hoarded in a functionalized nanoporous support via non-covalent interaction. Surprisingly, the protein released from the heated protein-nanoporous composite can maintain its nearly native conformation and activity, while free proteins are permanently denatured under the same treatment.

In vitro release of organophosphorus acid anhydrolase from functionalized mesoporous silica against nerve agents.

We report here that under different physiological conditions, biomolecular drugs can be stockpiled in a nanoporous support and afterward can be instantly released when needed for acute responses, and the biomolecular drug molecules can also be gradually released from the nanoporous support over a long time for a complete recovery. Organophosphorus acid anhydrolase (OPAA) was spontaneously and largely entrapped in functionalized mesoporous silica (FMS) due to the dominant electrostatic interaction. The OPAA-FMS composite exhibited a burst release in a pH 9.0 NaHCO₃-Na₂CO₃ buffer system and a gradual release in pH 7.4 simulated body fluid. The binding of OPAA to NH₂-FMS can result in less tyrosinyl and tryptophanyl exposure OPAA molecules to aqueous environment. The bound OPAA in FMS displayed lower activity than the free OPAA in solution prior to the enzyme entrapment. However, the released enzyme maintained the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. The in vitro results in the rabbit serum demonstrate that both OPAA-FMS and the released OPAA may be used as a medical countermeasure against the organophosphorus nerve agents.

Conformational Variability of Organophosphorus Hydrolase upon Soman and Paraoxon Binding

The bacterial enzyme organophosphorus hydrolase (OPH) exhibits both catalytic and substrate promiscuity. It hydrolyzes bonds in a variety of phosphotriester (P-O), phosphonothioate (P-S), phosphofluoridate (P-F), and phosphonocyanate (F-CN) compounds. However, its catalytic efficiency varies markedly for different substrates, limiting the broad-range application of OPH as catalyst in the bioremediation of pesticides and chemical war agents. In the present study, pK(a) calculations and multiple explicit-solvent molecular dynamics (MD) simulations were performed to characterize and contrast the structural dynamics of OPH bound to two substrates hydrolyzed with very distinct catalytic efficiencies: the nerve agent soman (O-pinacolylmethylphosphonofluoridate) and the pesticide paraoxon (diethyl p-nitrophenyl phosphate). pK(a) calculations for the substrate-bound and unbound enzyme showed a significant pK(a) shift from standard values (ΔpK(a) = ±3 units) for residues His254 and Arg275. MD simulations of protonated His254 revealed a dynamic hydrogen bond network connecting the catalytic residue Asp301 via His254 to Asp232, Asp233, Arg275, and Asp235, and is consistent with a previously postulated proton relay mechanism to ferry protons away from the active site with substrates that do not require activation of the leaving group. Hydrogen bonds between Asp301 and His254 were persistent in the OPH-paraoxon complex but not in the OPH-soman one, suggesting a potential role for such interaction in the more efficient hydrolysis of paraoxon over soman by OPH. These results are in line with previous mutational studies of residue His254, which led to an increase of the catalytic efficiency of OPH over soman yet decreased its efficiency for paraoxon. In addition, comparative analysis of the molecular trajectories for OPH bound to soman and paraoxon suggests that binding of the latter facilitates the conformational transition of OPH from the open to the closed substate promoting a tighter binding of paraoxon.

In situ regeneration of NADH via lipoamide dehydrogenase-catalyzed electron transfer reaction evidenced by spectroelectrochemistry.

NAD/NADH is a coenzyme found in all living cells, carrying electrons from one reaction to another. We report on characterizations of in situ regeneration of NADH via lipoamide dehydrogenase (LD)-catalyzed electron transfer reaction to regenerate NADH using UV-vis spectroelectrochemistry. The Michaelis-Menten constant (K(m)) and maximum velocity (V(max)) of NADH regeneration were measured as 0.80±0.15 mM and 1.91±0.09 μM s(-1) in a 1-mm thin-layer spectroelectrochemical cell using gold gauze as the working electrode at the applied potential -0.75 V (vs. Ag/AgCl). The electrocatalytic reduction of the NAD system was further coupled with the enzymatic conversion of pyruvate to lactate by lactate dehydrogenase to examine the coenzymatic activity of the regenerated NADH. Although the reproducible electrocatalytic reduction of NAD into NADH is known to be difficult compared to the electrocatalytic oxidation of NADH, our spectroelectrochemical results indicate that the in situ regeneration of NADH via LD-catalyzed electron transfer reaction is fast and sustainable and can be potentially applied to many NAD/NADH-dependent enzyme systems.

Single-molecule electron transfer reactions in nanomaterials

Here we report the study of single molecule electron transfer dynamics by coupling fluorescence microscopy at a conventional electrochemical cell. The single-molecule fluorescence spectroelectrochemistry of cresyl violet in aqueous solution and on nanoparticle surface were studied. We observed that the single-molecule fluorescence intensity of cresyl violet is modulated synchronously with the cyclic voltammetric potential scanning. We attribute the fluorescence intensity change of single cresyl violet molecules to the electron transfer reaction driven by the electrochemical potential.