Organophosphorus hydrolase (OPH) is a metalloenzyme that can hydrolyze organophosphorus agents resulting in products that are generally of reduced toxicity. The best OPH substrate found to date is diethyl p-nitrophenyl phosphate (paraoxon). Most structural and kinetic studies assume that the binding orientation of paraoxon is identical to that of diethyl 4-methylbenzylphosphonate, which is the only substrate analog co-crystallized with OPH. In the current work, we used a combined docking and molecular dynamics (MD) approach to predict the likely binding mode of paraoxon. Then, we used the predicted binding mode to run MD simulations on the wild type (WT) OPH complexed with paraoxon, and OPH mutants complexed with paraoxon. Additionally, we identified three hot-spot residues (D253, H254, and I255) involved in the stability of the OPH active site. We then experimentally assayed single and double mutants involving these residues for paraoxon binding affinity. The binding free energy calculations and the experimental kinetics of the reactions between each OPH mutant and paraoxon show that mutated forms D253E, D253E-H254R, and D253E-I255G exhibit enhanced substrate binding affinity over WT OPH. Interestingly, our experimental results show that the substrate binding affinity of the double mutant D253E-H254R increased by 19-fold compared to WT OPH.
Porphyrins are vital pigments involved in biological energy transduction processes. Their abilities to absorb light, then convert it to energy, have raised the interest of using porphyrin nanoparticles as photosensitizers in photodynamic therapy. A recent study showed that self- assembled porphyrin-silica composite nanoparticles can selectively destroy tumor cells, but detection of the cellular uptake of porphyrin-silica composite nanoparticles was limited to imaging microscopy. Here we developed a novel method to rapidly identify porphyrin-silica composite nanoparticles using Atmospheric Solids Analysis Probe-Mass Spectrometry (ASAP-MS). ASAP-MS can directly analyze complex mixtures without the need for sample preparation. Porphyrin-silica composite nanoparticles were vaporized using heated nitrogen desolvation gas, and their thermo-profiles were examined to identify distinct mass- to-charge (M/Z) signatures. HeLa cells were incubated in growth media containing the nanoparticles, and after sufficient washing to remove residual nanoparticles, the cell suspension was loaded onto the end of ASAP glass capillary probe. Upon heating, HeLa cells were degraded and porphyrin-silica composite nanoparticles were released. Vaporized nanoparticles were ionized and detected by MS. The cellular uptake of porphyrin-silica composite nanoparticles was identified using this ASAP-MS method.
Low dimensional lead halide perovskite particles are of tremendous interest due to their size-tunable band gaps, low exciton binding energy, high absorption coefficients, outstanding quantum and photovoltaic efficiencies. In this paper, we report a new solution-based synthesis of stabilized Cs4PbBr6 perovskite particles with high luminescence. This method requires only mild conditions and produces colloidal particles that are ideal for highly efficient solution-based device fabrications. The synthesized microstructures not only display outstanding luminescence quantum yield but also long term stability in atmospheric conditions. Partial halide substitutions were also demonstrated to extend photoluminescence spectra of the perovskite particles. Finally, this convenient synthesis and optical tunability of Cs4PbBr6 perovskite particles will be advantageous for future applications of optoelectronic advices.
In an effort to utilize their unique photoactive properties, porphyrin monomers were assembled into tetragonal microparticles by a surfactant-assisted neutralization method through the cooperative interactions between the porphyrin building blocks including π-π stacking, J-aggregation and metal-ligand coordination. Electron microscopy characterization in combination with X-ray diffraction confirmed the three-dimensional ordered tetragonal microstructures with stable crystalline frameworks and well defined external surface morphology. Optical absorption and fluorescence spectroscopy revealed enhanced absorbance properties as compared with the raw porphyrin material, favourable for chromophore excitation and energy transport. With active and responsive optical properties, these new porphyrin microparticles look to serve as promising components for a wide range of applications including sensing, diagnostics, solar cells, and optoelectronic devices.
We describe a new method to measure the activation energy for unbinding (enthalpy ΔH*u and free energy ΔG*u) of a strongly-bound membrane-associated protein from a lipid membrane. It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method is used to determine ΔH*u and ΔG*u for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH 5.5. ΔH*u is determined from the Arrhenius equation whereas ΔG*u is determined by fitting the data to a model based on mean first passage time for escape from a potential well. The binding free energy ΔGb of sE was also measured at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipid bilayer. The unbinding free energy (20 ± 3 kcal/mol, 20% PG) was found to be roughly three times the binding energy per monomer, (7.8 ± 0.3 kcal/mol for 30% PG, or est. 7.0 kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH 5.5, but assembles into trimers after associating with membranes. This new method to determine unbinding energies should be useful to understand better the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.
Here, we describe a new method to measure the activation energy required to remove a strongly-bound membrane-associated protein from a lipid membrane (anchoring energy). It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method was used to determine anchoring energy for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH 5.5. We also measured the binding energy of sE at the same pH for the initial, predominantly reversible, phase of binding to a 70:30 PC:PG lipid bilayer. The anchoring energy (37 +/- 1.7 kcal/mol, 20% PG) was found to be much larger than the binding energy (7.8 +/- 0.3 kcal/mol for 30% PG, or est. 7.0 kcal/mol for 20% PG). This is consistent with data showing that free sE is a monomer at pH 5.5, but assembles into trimers after associating with membranes. But, trimerization alone is insufficient to account for the observed difference in energies, and we conclude that some energy dissipation occurs during the release process. This new method to determine anchoring energy should be useful to understand the complex interactions of integral monotopic proteins and strongly-bound peripheral membrane proteins with lipid membranes.
Organophosphates are widely used for peaceful (agriculture) and military purposes (chemical warfare agents). The extraordinary toxicity of organophosphates and the risk of deployment, make it critical to develop means for their rapid and efficient deactivation. Organophosphate hydrolase (OPH) already plays an important role in organophosphate remediation, but is insufficient for therapeutic or prophylactic purposes primarily due to low substrate affinity. Current efforts focus on directly modifying the active site to differentiate substrate specificity and increase catalytic activity. Here, we present a novel strategy for enhancing the general catalytic efficiency of OPH through computational redesign of the residues that are allosterically coupled to the active site and validated our design by mutagenesis. Specifically, we identify five such hot-spot residues for allosteric regulation and assay these mutants for hydrolysis activity against paraoxon, a chemical-weapons simulant. A high percentage of the predicted mutants exhibit enhanced activity over wild-type (kcat =16.63 s-1), such as T199I/T54I (899.5 s-1) and C227V/T199I/T54I (848 s-1), while the Km remains relatively unchanged in our high-throughput cell-free expression system. Further computational studies of protein dynamics reveal four distinct distal regions coupled to the active site that display significant changes in conformation dynamics upon these identified mutations. These results validate a computational design method that is both efficient and easily adapted as a general procedure for enzymatic enhancement.