Active Site Contacts Research Articles

Flavonoid-Quinoxaline Hybrid Compounds as Cathepsin Inhibitors Against Fascioliasis.

Fasciola hepatica is a parasitic trematode that infects livestock animals and humans, causing significant health and economic burdens worldwide. The extensive use of anthelmintic drugs has led to the emergence of resistant parasite strains, posing a threat to treatment success. The complex life cycle of the liver fluke, coupled with limited funding and research interest, have hindered progress in drug discovery. Our group has been working in drug development against this parasite using cathepsin proteases as molecular targets, finding promising compound candidates with in vitro and in vivo efficacy. Here, we evaluated hybrid molecules that combine two chemotypes, chalcones and quinoxaline 1,4-di- N-oxides, previously found to inhibit F. hepatica cathepsin Ls and tested their in vitro activity with the isolated targets and the parasites in culture. These molecules proved to be good cathepsin inhibitors and to kill the juvenile parasites at micromolar concentrations. Also, we performed molecular docking studies to analyze the compounds-cathepsins interface, finding that the best inhibitors interact at the active site cleft and contact the catalytic dyad and residues belonging to the substrate binding pockets. We conclude that the hybrid compounds constitute promising scaffolds for the further development of new fasciolicidal compounds.

Dependence of the strong metal-support interactions of Ni/TiO2 and the induction period of glycerol steam reforming on the dynamic crystal phase evolution

The dynamic relationship of TiO2 crystal phase and strong metal-support interactions (SMSI) of Ni/TiO2 catalyst was investigated to reveal the induction period of glycerol steam reforming. Comprehensive investigations confirmed that the SMSI between Ni0 and anatase TiO2 was formed by anatase TiO2 encapsulated Ni0 after H2 reduction at 500 °C for the Ni/TiO2 catalyst calcined at 500 °C (Ni/Ti-500R), which inhibited the contact of Ni0 active sites with reactants and led to the low hydrogen yield in the initial stage of the reaction. Interestingly, the dynamic conversion of anatase to rutile during the GSR process at 600 °C caused the TiO2 overlayers on the Ni0 particles to gradually fade, exposing more Ni0 sites, resulting in a gradual increase in hydrogen yield to 5.93 molH2/molglycerol. In addition, Ni/Ti-500R with a dynamic SMSI inhibited Ni0 particles sintering and encapsulated carbon deposition, resulting in relatively better stability (21 h) after induction period. In comparison, the designed Ni/TiO2 catalyst calcined at 600 °C (Ni/Ti-600R) with rutile TiO2 as the main component inhibited the formation of the classical SMSI, even after 600 °C H2 reduction, and no excessive TiO2 encapsulated Ni0 particles were observed, so no obvious induction period occurred. This study provides new insights into the relationship of SMSI with crystal phases and the induction period in multiphase catalysis, also a new strategy for designing efficient catalysts.

Enzyme-free glucose sensor based on electrodeposition of multi-walled carbon nanotubes and Zn-based metal framework-modified gold electrode at low potential

A non-enzymatic glucose sensor was successfully prepared by the electrodeposition methods assembling of Zn-metal-organic frameworks/multi-walled carbon nanotubes (Zn-MOF/MWCNTs) on gold electrode (GE). The sensor showed better catalytic oxidation performance for glucose at very low potential (0.20 V) compared to the bare GE (0.30 V), because the modified complex can effectively increase the active site and reaction contact area. The sensor exhibited a wider linear detection range of 0.020–8.14 mM with a detection limit of 0.037 mΜ. In addition, the electrochemical response of the sensor to glucose showed good selectivity, which was further verified in the complex serum environment by the standard addition recovery method. The recovery of the sensor was in the range of 98.52–102.65%, with a relative standard deviation (RSD) of no more than 8.00%, showing the potential applications in real testing.

Renewable Hydrogen Production with Steam Reforming of Ethanol Using Siliceous Mesocellular Foam‐Supported Nickel Catalysts

Nickel (Ni) catalysts loaded on siliceous mesocellular foam (MCF‐S) are synthesized via the wet impregnation method with 3, 5, 10, and 15 wt% NiO loadings and different aging levels (no, partial, and full ageing) to determine how both factors affect the progress of the ethanol steam reforming (ESR) reaction. After extensive material characterization testing to determine material porosity, crystallinity, and Ni metal particle size and spatial location, as well as reaction testing at 300–700 °C and 4 H2O: 1 EtOH molar ratio, the fully aged 10 wt% Ni/MCF‐S possesses the strongest structural stability and catalytic activity, reaching 100% EtOH conversion and 68% H2 selectivity at 700 °C. The aging disperses and embeds more Ni nanoparticles within the walls of the mesopores, which promote the ESR reaction from easier diffusion and more active site contact within the pores. Furthermore, the catalyst reveals little signs of deactivation, as the structure remains virtually unchanged, and any coke formed is on the silica support and not over the Ni nanoparticles after the ESR reaction. Such results have demonstrated a proven applicability for ESR and a further need to research about aging effects toward improving structural properties and the catalytic reaction activity.

Evaluating the active site-substrate interplay between x-ray crystal structure and molecular dynamics in chorismate mutase.

Designing realistic quantum mechanical (QM) models of enzymes is dependent on reliably discerning and modeling residues, solvents, and cofactors important in crafting the active site microenvironment. Interatomic van der Waals contacts have previously demonstrated usefulness toward designing QM-models, but their measured values (and subsequent residue importance rankings) are expected to be influenceable by subtle changes in protein structure. Using chorismate mutase as a case study, this work examines the differences in ligand-residue interatomic contacts between an x-ray crystal structure and structures from a molecular dynamics simulation. Select structures are further analyzed using symmetry adapted perturbation theory to compute ab initio ligand-residue interaction energies. The findings of this study show that ligand-residue interatomic contacts measured for an x-ray crystal structure are not predictive of active site contacts from a sampling of molecular dynamics frames. In addition, the variability in interatomic contacts among structures is not correlated with variability in interaction energies. However, the results spotlight using interaction energies to characterize and rank residue importance in future computational enzymology workflows.

High-Surface-Area Metalloporphyrin-Based Porous Ionic Polymers by the Direct Condensation Strategy for Enhanced CO2 Capture and Catalytic Conversion into Cyclic Carbonates.

Metalloporphyrin-based porous organic polymers (POPs) that behave as advanced biomimetic nanoreactors have drawn continuous attention for heterogeneous CO2 catalysis in the past decades. Inspired by the double activation model of epoxides, the design and synthesis of metalloporphyrin-based porous ionic polymers (PIPs) are considered as one of the most promising approaches for converting CO2 to cyclic carbonates under cocatalyst- and solvent-free conditions. To overcome the obstacle of poor reaction activity of ionic monomers or highly irregular stacking architecture, in this paper, we have proposed and demonstrated a modular bottom-up approach for constructing a series of high-surface-area metalloporphyrin-based PIPs in high yields by the direct condensation strategy, thus boosting the close contact of multiple active sites and achieving the enhanced CO2 capture and catalytic conversion into cyclic carbonates with high turnover frequencies under mild conditions. These recyclable aluminum-porphyrin-based PIPs are featured with high surface areas, prominent CO2 adsorptive capacities, rigid porphyrin skeletons, and flexible ionic pendants, as well as the matched amounts and spatial positions of metal centers and ionic sites, in which is demonstrated to be one of the quite competitive catalysts. Therefore, this strategy of introducing ionic components into the porphyrin frameworks as flexible side chains rather than main chains and adjusting the reactivity ratios of comonomers by structure-oriented methods, provides feasible guidance for the multifunctionalization of metalloporphyrin-based POPs, thereby increasing the accessibility of multiple active sites and improving their synergistic catalytic behavior.

Enhanced catalytic activity of Fe–Ce catalysts determined by synergistic effect of different precursors in selective catalytic reduction of NOx with NH3

AbstractBackgroundDeveloping environmentally benign and non‐vanadium SCR catalysts remains a challenging task for NOx abatement. In this article, Fe–Ce catalysts including Fe–Ce(S) and Fe(S)–Ce catalysts with different molar ratios of Fe/Ce were synthesized by different precursors of active species expecting to gain high catalytic activity for the selective catalytic reduction (SCR) of NOx with NH3 in a wide temperature range.ResultsThe Fe–Ce(S) catalyst exhibited better catalytic activity than the Fe(S)–Ce catalyst. The characterization results indicated the Fe–Ce(S)catalyst possessed better dispersion of active species, which would favor the contact of active sites and reactant molecules and thus enhance the catalytic performance. Moreover, higher ratios of Fe3+ and Ce3+ and more surface chemisorbed oxygen were observed over the Fe–Ce(S) catalyst. In addition, the Fe–Ce(S) catalyst held better low‐temperature redox properties and more weak acid sites.ConclusionsThe precursor effect was closely related to the catalytic activity of catalysts. All of the mentioned above might be the main reasons for the superior catalytic performance of the Fe–Ce(S) catalyst, especially at low temperatures. These findings indicate that Fe–Ce(S) catalyst is a promising catalyst for the SCR reaction to replace V‐W‐Ti catalysts and can provide guidance for the development of environmentally benign and non‐vanadium SCR catalysts. © 2022 Society of Chemical Industry (SCI).

Triple-Phase Interface Engineered Hierarchical Porous Electrode for CO2 Electroreduction to Formate.

The aqueous electrochemical CO2 reduction to valuable products is seen as one of the most promising candidates to achieve carbon neutrality yet still suffers from poor selectivity and lower current density. Highly efficient CO2 reduction significantly relies on well‐constructed electrode to realize efficient and stable triple‐phase contact of CO2, electrolyte, and active sites. Herein, a triple‐phase interface engineering approach featuring the combination of hierarchical porous morphology design and surface modification is presented. A hierarchical porous electrode is constructed by depositing bismuth nanosheet array on copper foam followed by trimethoxy (1H,1H,2H,2H‐heptadecafluorodecyl) silane modification on the nanosheet surface. This electrode not only achieves highly selective and efficient CO2 reduction performance with formate selectivity above 90% over wide potentials and a partial current density over −90 mA cm−2 in H‐cell but also maintains a superior stability during the long‐term operation. It is demonstrated that this remarkable performance is attributed to the construction of efficient and stable triple‐phase interface. Theoretical calculations also show that the modified surface optimizes the activation path by lowering thermodynamic barriers of the key intermediates *OCHO for the formation of formate during electrochemical CO2 reduction.

Evolution of Triple-Phase interface for enhanced electrochemical CO2 reduction

Selective electrochemical CO2 reduction reaction (CO2RR) requires efficient triple-phase contact of CO2 molecules, electrolyte and active sites of electrocatalyst. To achieve this, studies have sought to use surface modifications to electrodes. However, the stability of these modifications and their effects on the intrinsic properties of electrodes are rarely discussed. Here, hydrophobic treatment of 1-octadecanethiol modification to a copper dendrite electrode was taken as a benchmark to investigate the evolution of the electrode surface and corresponding CO2RR performance after modifications. We show that the initial hydrophobic surface (HB-Cu) eventually transformed into a stable triple-phase interface (TP-Cu), ascribed to the formation of stable copper thiolate and the generation of crevices from alkane sulfonate dissolution. Surprisingly, this structural evolution leads to lower resistance of charge transfer and more sufficient exposure of active sites, enabling efficient triple-phase contact. As a result, TP-Cu achieved a near 4-fold enhancement of current density and a more than 2-fold reduction of H2 production compared to HB-Cu. Compared to previous studies, we have demonstrated extra power of the surface modification strategy to design triple-phase electrode interface, facilitating selective and efficient electrochemical CO2 reduction.

Evolution of surface acidity during smectite illitization: Implication for organic carbon cycle

The clay-organic interactions in marine sediments and mudstones have long been concerned and play key roles in preservation of organic matter (OM). As chemically active mineral, smectite could transform into illite naturally, however, how this process affects stability and cycle of OM remains unclear. Here, we find that solid acidity varies greatly in terms of amount, type, and strength during illitization. The amount and strength of Brønsted (B) acid sites are enhanced markedly and interlayer space remains expandable in randomly interstratified illite/smectite, which enables immediate contact of active acid sites and adsorbed OM. In this case, the promotion of clay minerals on OM evolution would be greatly enhanced. Different from the traditional views on the effects of clay-OM interactions on organic carbon cycle, which include adsorptive protection and desorption, a new notion of clay mineral reactor is proposed which allows in-situ decomposition or degradation of OM.

Assembly of a dsRNA synthesizing complex: RNA-DEPENDENT RNA POLYMERASE 2 contacts the largest subunit of NUCLEAR RNA POLYMERASE IV

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV's 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3' ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs).

Hierarchical Ti-Beta zeolites with uniform intracrystalline mesopores hydrothermally synthesized via interzeolite transformation for oxidative desulfurization

Precisely designing and constructing metallosilicate zeolite with abundant and uniform intracrystalline mesopore is urgently desired, bearing the merits of boosting accessibility of active sites and of improving diffusion properties for organics in the process of catalytic reactions. Herein, hierarchical Ti-Beta zeolite with uniform intracrystalline mesopore (ca. 3.4 nm) is hydrothermally synthesized via interzeolite transformation of siliceous FAU zeolite, which is not capable to be achieved using amorphous silica sources. The crystallization process is thoroughly explored via series of characterization methods, obeying liquid crystallization mechanism. Additionally, the assembly of hierarchical Ti-Beta zeolite is nourished by the primary structural units (mainly 4-membered ring) resulting from the degraded silicate species of FAU zeolite. This burgeoning hierarchical Ti-Beta zeolite shows extraordinary catalytic performances in terms of both active site-based turnover frequency and weight-based space-time-yield for oxidative desulfurization of model oil, far beyond than conventional fluoride-mediated Ti-Beta and microporous Ti-Beta prepared by this developed strategy without addition of CTAB, which is attributed to its excellent diffusion properties and undemanding contact of active sites with reaction substrates. This work provides a span-new guidance for designing hierarchical metallosilicates with uniform mesopore from the standpoint of employing exotic primary structural units derived from the degradation of crystallized siliceous zeolites.

Preparation of Nickel-Decorated and Nitrogen-Doped Carbon Nanotubes for Highly Efficient Hydrogen Evolution Reaction by Hydrothermal Method

The unique structure of nickel-decorated and nitrogen-doped carbon nanotubes (Ni-NCNTs) was prepared by hydrothermal method. Nickel acetate tetrahydrate, graphitic carbon nitride (g-C3N4), and isopropanol were used as raw materials in this moderate method. The Ni-NCNTs were synthesized successfully by calcination under the protection of nitrogen. Under different pyrolysis temperatures, carbon nanotubes begin to form at 700 °C, and Ni-NCNTs also show the best hydrogen evolution reaction performance. At a current density of 10 mA/cm2, the overpotential has reached 378 mV and the Tafel slope of the sample is 139 mV/dec. This work successfully presented that it is feasible to introduce Ni nanoparticles and nitrogen into CNTs to enhance the electrocatalytic performance, while the doping of transition metals, Ni, and nitrogen improves the conductivity of CNTs and increases the contact of active sites.

Palladium Nanoparticles Embedded in Yolk–Shell N-Doped Carbon Nanosphere@Void@SnO2 Composite Nanoparticles for the Photocatalytic Reduction of 4-Nitrophenol

Herein, a novel visible-light-driven yolk–shell nanoreactor has been fabricated, called as Pd/N–Cs@SnO2, which is consisted of movable nitrogen-doped carbon sphere (N–Cs) yolk, interlayer void, mesoporous tin oxide (SnO2) shell, and encapsulated palladium nanoparticles (Pd NPs). The obtained yolk–shell Pd/N–Cs@SnO2 nanoreactor can serve as an excellent catalyst in the visible-light-driven photocatalytic reduction of 4-nitrophenol (4-NP). More importantly, Pd/N–Cs@SnO2 exhibits a remarkable superior activity and higher reusability than core–shell Pd@SnO2 and yolk–shell Pd/N–Cs@mSiO2 nanoreactors. The fabulous character of Pd/N–Cs@SnO2 nanoreactor is attributed to (I) the mesostructured SnO2 shell provides a large surface area for the collection of light as well as the anchoring of Pd to avoid aggregating or leaching; (II) the interlayer void space improves the light absorption by multiple scattering, as well as favors the contact of active sites with reactants in the photocatalytic reactions; (III) the N–Cs allow more permeable visible-light absorption, and accelerate the photoexcited electrons transportation from SnO2 semiconductor to Pd NPs to suppress the electron–hole recombination. This enriches the electron density of Pd NPs surface and then facilitates the photocatalytic reduction of 4-NP. Both the structural advantages and enhanced synergy of Pd/N–Cs@SnO2 nanoreactor make it to be an efficient and stable photocatalyst, which can open an avenue for designing photocatalysts in a board range of organic reactions.

Using NMR Derived Structural Information Complemented by Molecular Docking and MM/GBSA Rescoring to Mitigate CYP3A4 Time‐Dependent Inhibition

Time‐dependent inhibition (TDI) of human CYP3A4 is a significant challenge in drug development and is the cause of multiple clinically relevant drug‐drug interactions. The vasodilator diltiazem causes TDI when metabolized by CYP3A4. We hypothesized that introduction of steric bulk into the CYP3A4 active site by site‐directed mutagenesis will disrupt active site contacts, alter diltiazem orientation in the CYP3A4 active site, and decrease TDI, as previously seen with midazolam. CYP3A4‐ligand interactions were monitored using spectral binding titrations. In parallel, we performed molecular docking of diltiazem and the N‐demethylation products into CYP3A4 models of previously tested mutants that introduce steric bulk into the enzyme active site, followed by molecular mechanics generalized Born surface area (MM‐GBSA) rescoring. An NMR‐derived model of diltiazem in the CYP3A4 active site revealed that the dimethylamino group that undergoes CYP3A4‐mediated N‐demethylation is oriented toward the heme iron. Diltiazem caused a Type II spin shift upon binding to CYP3A4, indicating interaction of the amine with the heme iron. Docking of diltiazem, N‐desmethyldiltiazem, or N, N‐didesmethyldiltiazem into wild‐type CYP3A4 resulted in the amino group of each compound oriented toward the heme iron. In contrast, docking these three compounds into CYP3A4 I120W resulted in the amino group orienting away from the heme. Furthermore, T309Y docking results showed reorientation of diltiazem, which would prevent the initial N‐demethylation event, and I301W and A370F resulted in N‐desmethyldiltiazem reorientation. MM‐GBSA rescoring of docking experiments confirmed that all compounds used orient with the amino group toward the heme in wild‐type CYP3A4, all amino groups away from the heme in I120W, and reorientation of N‐desmethyldiltiazem in I301W and A370F. Furthermore, in rescoring experiments, N‐desmethyldiltiazem was reoriented in the Y307W active site, and N, N‐didesmethyldiltiazem was reoriented in the T309Y active site in addition to reorientation of diltiazem. Substitution of I120 and T309 with a larger residue decreased CYP3A4 TDI. In summary, a hybrid NMR‐computational approach was used to rapidly generate a model of diltiazem in complex with CYP3A4. These results indicate that NMR‐derived models of protein‐ligand interactions provide a structure‐based approach for decreasing metabolic liabilities of CYP enzyme substrates.Support or Funding InformationThis work was supported by internal funding from the University of Connecticut to James Halpert.

Using CYP3A4 NMR derived structural information to mitigate time‐dependent inhibition due to diltiazem metabolism

Human CYP3A4 metabolizes a significant fraction of clinically relevant drugs. CYP3A4‐mediated metabolism of the vasodilator diltiazem leads to time‐dependent inhibition (TDI) and clinically relevant drug‐drug interactions. We hypothesized that introduction of steric bulk into the CYP3A4 active site by site‐directed mutagenesis will disrupt active site contacts, alter diltiazem orientation in the CYP3A4 active site, and decrease TDI, as previously seen with midazolam. A model of diltiazem in the CYP3A4 active site was produced using distance restraints generated from longitudinal T1 NMR relaxation experiments. CYP3A4‐ligand interactions were also monitored using spectral binding titrations. Evaluation of diltiazem‐mediated CYP3A4 TDI was performed using the reporter substrate 7‐ethoxy‐4‐(trifluoromethyl)coumarin. The diltiazem model revealed that the dimethylamino group that undergoes CYP3A4‐mediated N‐demethylation is oriented toward the heme iron with the aromatic ring of the benzothiazepine core near active site residues I369 and L373, the ester moiety near L216, and the p‐hydroxyphenyl group near S119. Diltiazem caused a Type II spin shift upon binding to CYP3A4, indicating interaction of the amine with the heme iron. Substitution of I369 with a larger residue decreased CYP3A4 TDI, and an NMR derived model of diltiazem in complex with CYP3A4 I369W revealed that the p‐hydroxyphenyl group was toward the heme iron instead of the dimethylamino group. In summary, a hybrid NMR‐computational approach was used to rapidly generate a model of diltiazem in complex with CYP3A4. Introduction of steric bulk into the CYP3A4 active site by mutagenesis altered the orientation of diltiazem and mitigated TDI. The next step is to add steric bulk to the cognate part of the compound. These results indicate that NMR‐derived models of protein‐ligand interactions provide a structure‐based approach for decreasing metabolic liabilities of CYP enzyme substrates.Support or Funding InformationThis work was supported by internal funding from the University of Connecticut to James Halpert.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase.

Human 5'-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5'-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (-ΔCp), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔCp of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔCp. Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔCp for binding. In addition, ΔCp values are not correlated with Kd values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (-ΔCp‡). A comparison of the ΔCp‡ for MTAP presteady state chemistry and ΔCp for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP-inhibitor complexes indicate remarkable protein thermal stability (to Tm = 99 °C) in complexes with transition-state analogues.

Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics.

We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker than PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.

Luminescent Ru(II) Phenanthroline Complexes as a Probe for Real-Time Imaging of Aβ Self-Aggregation and Therapeutic Applications in Alzheimer's Disease.

The complexes cis-[Ru(phen)2(Apy)2]2+, Apy = 4-aminopyridine and 3,4-aminopyridine, are stable in aqueous solution with strong visible absorption. They present emission in the visible region with long lifetime that accumulates in the cytoplasm of Neuro2A cell line without appreciable cytotoxicity. The complexes also serve as mixed-type reversible inhibitors of human AChE and BuChE with high active site contact. cis-[Ru(phen)2(3,4Apy)2]2+ competes efficiently with DMPO by the OH• radical. Luminescence using fluorescence lifetime imaging (FLIM) enables real-time imaging of the conformational changes of the self-aggregation of Aβ with incubation of complexes (0-24 h) in phosphate buffer at micromolar concentrations. By this technique, we identified protofibrills in the self-assembly of Aβ1-40 and globular structures in the short fragment Aβ15-21 in aqueous solution.

Increased Turnover at Limiting O2 Concentrations by the Thr(387) → Ala Variant of HIF-Prolyl Hydroxylase PHD2.

PHD2 is a 2-oxoglutarate, non-heme Fe(2+)-dependent oxygenase that senses O2 levels in human cells by hydroxylating two prolyl residues in the oxygen-dependent degradation domain (ODD) of HIF1α. Identifying the active site contacts that determine the rate of reaction at limiting O2 concentrations is crucial for understanding how this enzyme senses pO2 and may suggest methods for chemically altering hypoxia responses. A hydrogen bonding network extends from the Fe(II) cofactor through ordered waters to the Thr(387) residue in the second coordination sphere. Here we tested the impact of the side chain of Thr(387) on the reactivity of PHD2 toward O2 through a combination of point mutagenesis, steady state kinetic experiments and {FeNO}(7) EPR spectroscopy. The steady state kinetic parameters for Thr(387) → Asn were very similar to those of wild-type (WT) PHD2, but kcat and kcat/KM(O2) for Thr(387) → Ala were increased by roughly 15-fold. X-Band electron paramagnetic resonance spectroscopy of the {FeNO}(7) centers of the (Fe+NO+2OG) enzyme forms showed the presence of a more rhombic line shape in Thr(387) → Ala than in WT PHD2, indicating an altered conformation for bound gas in this variant. Here we show that the side chain of residue Thr(387) plays a significant role in determining the rate of turnover by PHD2 at low O2 concentrations.