Green MIP-202(Zr) Catalyst: Degradation and Thermally Robust Biomimetic Sensing of Nerve Agents.

Rapid and robust sensing of nerve agent (NA) threats is necessary for real-time field detection to facilitate timely countermeasures. Unlike conventional phosphotriesterases employed for biocatalytic NA detection, this work describes the use of a new, green, thermally stable, and biocompatible zirconium metal-organic framework (Zr-MOF) catalyst, MIP-202(Zr). The biomimetic Zr-MOF-based catalytic NA recognition layer was coupled with a solid-contact fluoride ion-selective electrode (F-ISE) transducer, for potentiometric detection of diisopropylfluorophosphate (DFP), a F-containing G-type NA simulant. Catalytic DFP degradation by MIP-202(Zr) was evaluated and compared to the established UiO-66-NH2 catalyst. The efficient catalytic DFP degradation with MIP-202(Zr) at near-neutral pH was validated by 31P NMR and FT-IR spectroscopy and potentiometric F-ISE and pH-ISE measurements. Activation of MIP-202(Zr) using Soxhlet extraction improved the DFP conversion rate and afforded a 2.64-fold improvement in total percent conversion over UiO-66-NH2. The exceptional thermal and storage stability of the MIP-202/F-ISE sensor paves the way toward remote/wearable field detection of G-type NAs in real-world environments. Overall, the green, sustainable, highly scalable, and biocompatible nature of MIP-202(Zr) suggests the unexploited scope of such MOF catalysts for on-body sensing applications toward rapid on-site detection and detoxification of NA threats.

Determination of camostat and its metabolites in human plasma - Preservation of samples and quantification by a validated UHPLC-MS/MS method.

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

Mixed-Metal Cerium/Zirconium MOFs with Improved Nerve Agent Detoxification Properties.

A series of Ce/Zr mixed-metal-organic frameworks with different topology/connectivity, namely, Ce/Zr-UiO-66 (U01, U02, and U03) (fcu (12-c)), Ce/Zr-DUT-67-PZDC (D01 and D02) (reo (8-c)), and Ce/Zr-MOF-808 (M01, M02, and M03) (spn (6-c)) were evaluated toward the detoxification of toxic nerve agent model diisopropylfluorophosphate (DIFP) at room temperature in unbuffered aqueous solution. Noteworthily, the catalytic rate for P-F bond cleavage increased with increasing Ce/Zr molar ratio. A further increase in catalytic activity can be achieved by Mg(OMe)2 doping of the mixed-metal MOFs as exemplified with M01@Mg(OMe)2 and M02@Mg(OMe)2 systems. The results show that Mg(OMe)2 incorporation into the mesoporous cavities of M01 and M02 give rise to P-F hydrolytic degradation half-lives of nearly 5 and 2 min with 100% degradation of DIFP after 55 and 65 min for M01@Mg(OMe)2 1:2 and M02@Mg(OMe)2 1:4, respectively.

Sorption of G-agent simulant vapours on human scalp hair.

Human scalp hair is a biological matrix that can trap chemical vapours from explosives (TNT), drugs (THC) and chemical weapons (yperite). The external contamination of human's hair following exposure to organophosphorus (OP) nerve agent was simulated by model compounds: triethyl phosphate (TEP) and diisopropyl fluorophosphate (DFP). In this work were exposed strands of hair to vapours of TEP and DFP (3 and 7 ppmv) to model sorption kinetics. Sorption isotherms were also investigated at several contamination levels (80-3000 mg min.m-3). OP nerve agent simulants were extracted from hair by soaking in DCM. Raw extracts were analysed in GC-MS/MS to quantify each simulant content in hair. Results were fitted by applying isotherm or kinetic equations. The best model was found to be bimodal first-order, suggesting the co-existence of two different mechanisms of sorption. The best equation to describe OP vapours incorporation on hair was Freundlich model. Thus hair can be used as a passive sensor able to trap chemical G-agents and can also offer valuable information regarding both individual contamination and proof of exposure to chemical weapons.

Structural and kinetic evidence of aging after organophosphate inhibition of human Cathepsin A.

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue, SCPEP1, as potential targets of organophosphates (OP). CatA could be stably inhibited by low µM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (KI) of 2 µM followed by VR (KI = 2.8 µM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 × 103 M-1sec-1 and 1.2 × 103 M-1sec-1, respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had KIDFP = 13 µM and 11 µM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a "backdoor" for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP.

Engineering Dynamic Surface Peptide Networks on ButyrylcholinesteraseG117H for Enhanced Organophosphosphorus Anticholinesterase Catalysis.

The single residue mutation of butyrylcholinesterase (BChEG117H) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in KM and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.

Two phase I, pharmacokinetic, and pharmacodynamic studies of DFP-10917, a novel nucleoside analog with 14-day and 7-day continuous infusion schedules.

Purpose DFP-10917 is a novel deoxycytidine analog with a unique mechanism of action. Brief exposure to high concentrations of DFP-10917 inhibits DNA polymerase resulting in S-phase arrest, while prolonged exposure to DFP-10917 at low concentration causes DNA fragmentation, G2/M-phase arrest, and apoptosis. DFP-10917 demonstrated activity in tumor xenografts resistant to other deoxycytidine analogs. Experimental design Two phase I studies assessed the safety, pharmacokinetic, pharmacodynamic and preliminary efficacy of DFP-10917. Patients with refractory solid tumors received DFP-10917 continuous infusion 14-day on/7-day off and 7-day on/7-day off. Enrollment required age > 18 years, ECOG Performance Status 0-2 and adequate organ function. Results 29 patients were dosed in both studies. In 14-day infusion, dose-limiting toxicities (DLT) consisting of febrile neutropenia and thrombocytopenia occurred at 4.0 mg/m2/day. At 3.0 mg/m2/day, 3 patients experienced neutropenia in cycle 2. The dose of 2.0 mg/m2/day was well tolerated in 6 patients. In 7-day infusion, grade 4 neutropenia was DLT at 4.0 mg/m2/day. The maximum tolerated dose was 3 mg/m2/day. Other toxicities included nausea, vomiting, diarrhea, neutropenia, and alopecia. Eight patients had stable disease for >12 weeks. Paired comet assays performed for 7 patients showed an increase in DNA strand breaks at day 8. Pharmacokinetic data showed dose-proportionality for steady-state concentration and AUC of DFP-10917 and its primary metabolite. Conclusion Continuous infusion of DFP-10917 is feasible and well tolerated with myelosuppression as main DLT. The recommended doses are 2.0 mg/m2/day and 3.0 mg/m2/day on the 14-day and 7-day continuous infusion schedules, respectively. Preliminary activity was suggested. Pharmacodynamic data demonstrate biological activity at the tested doses.

Acetylcholinesterase-capped Mesoporous Silica Nanoparticles Controlled by the Presence of Inhibitors.

Two different acetylcholinesterase (AChE)-capped mesoporous silica nanoparticles (MSNs), S1-AChE and S2-AChE, were prepared and characterized. MSNs were loaded with rhodamine B and the external surface was functionalized with either pyridostigmine derivative P1 (to yield solid S1) or neostigmine derivative P2 (to obtain S2). The final capped materials were obtained by coordinating grafted P1 or P2 with AChE's active sites (to give S1-AChE and S2-AChE, respectively). Both materials were able to release rhodamine B in the presence of diisopropylfluorophosphate (DFP) or neostigmine in a concentration-dependent manner via the competitive displacement of AChE through DFP and neostigmine coordination with the AChE's active sites. The responses of S1-AChE and S2-AChE were also tested with other enzyme inhibitors and substrates. These studies suggest that S1-AChE nanoparticles can be used for the selective detection of nerve agent simulant DFP and paraoxon.

Resolving pathways of interaction of mipafox and a sarin analog with human acetylcholinesterase by kinetics, mass spectrometry and molecular modeling approaches.

The hydroxyl oxygen of the catalytic triad serine in the active center of serine hydrolase acetylcholinesterase (AChE) attacks organophosphorus compounds (OPs) at the phosphorus atom to displace the primary leaving group and to form a covalent bond. Inhibited AChE can be reactivated by cleavage of the Ser-phosphorus bond either spontaneously or through a reaction with nucleophilic agents, such as oximes. At the same time, the inhibited AChE adduct can lose part of the molecule by progressive dealkylation over time in a process called aging. Reactivation of the aged enzyme has not yet been demonstrated. Here, our goal was to study oxime reactivation and aging reactions of human AChE inhibited by mipafox or a sarin analog (Flu-MPs, fluorescent methylphosphonate). Progressive reactivation was observed after Flu-MPs inhibition using oxime 2-PAM. However, no reactivation was observed after mipafox inhibition with 2-PAM or the more potent oximes used. A peptide fingerprinted mass spectrometry (MS) method, which clearly distinguished the peptide with the active serine (active center peptide, ACP) of the human AChE adducted with OPs, was developed by MALDI-TOF and MALDI-TOF/TOF. The ACP was detected with a diethyl-phosphorylated adduct after paraoxon inhibition, and with an isopropylmethyl-phosphonylated and a methyl-phosphonylated adduct after Flu-MPs inhibition and subsequent aging. Nevertheless, nonaged nonreactivated complexes were seen after mipafox inhibition and incubation with oximes, where MS data showed an ACP with an NN diisopropyl phosphoryl adduct. The kinetic experiments showed no reactivation of activity. The computational molecular model analysis of the mipafox-inhibited hAChE plots of energy versus distance between the atoms separated by dealkylation showed a high energy demand, thus little aging probability. However, with Flu-MPs and DFP, where aging was observed in our MS data and in previously published crystal structures, the energy demand calculated in modeling was lower and, consequently, aging appeared as a more likely reaction. We document here direct evidence for a phosphorylated hAChE refractory to oxime reactivation, although we observed no aging.

[COMPARATIVE SENSITIVITY OF CHOLINESTERASES IN VERTEBRATES AND INVERTEBRATES TO HIGHLY SPECIFIC ORGANOPHOSPHORUS INHIBITORS DIISOPROPYL FLUOROPHOSPHATE (DFP) AND (2-ETHOXYMETHYL PHOSPHORYL THIOETHYL) ETHYL (METHYL) SULPHONIUM SULPHOMETHYLAT (GD-42)].

The review presents data on comparative reactivity of 68 cholinesterase preparation from various organs and tissues in a number of vertebrates and invertebrates based on sensitivity to two highly specific and most studied organophosphorus inhibitors--diisopropyl fluorophosphates (DFP) and (2-ethoxymethyl phosphoryl thioethyl) ethyl (methyl) sulphonium sulphomethylat (GD-42). Analysis of these data suggests a great diversity in enzymologic characteristics of cholinesterase preparation in representatives of vertebrates and invertebrates, this variety observed even for closely related enzymes in animals of almost the same level of development.

Discovery of non-oxime reactivators using an in silico pharmacophore model of reactivators for DFP-inhibited acetylcholinesterase.

Utilizing our previously reported in silico pharmacophore model for reactivation efficacy of oximes, we present here a discovery of twelve new non-oxime reactivators of diisopropylfluorophosphate (DFP)-inhibited acetylcholinesterase (AChE) obtained through virtual screening of an in-house compound database. Rate constant (kr) efficacy values of the non-oximes were found to be within ten-fold of pralidoxime (2-PAM) in an in vitro DFP inhibited eel AChE assay and one of them showed in vivo efficacy comparable to 2-PAM against brain symptoms for DFP induced neuropathology in guinea pigs. Short listing of the identified compounds were performed on the basis of in silico evaluations for favorable blood brain barrier penetrability, octanol-water partition (Clog P), toxicity (rat oral LD50) and binding affinity to the active site of the crystal structure of a OP- inhibited AChE.

Theoretical and experimental investigation of photophysical properties of Zn(DFP SAMQ)2.

Theoretical calculations and experimental measurements were carried out for the investigation of spectroscopic and photophysical properties of Zn(DFP SAMQ)2 complex. The rate constant of intersystem crossing and the radiative rate constant were calculated using ab initio method. The rate constant of the internal conversion was estimated using the received calculated values and the experimental fluorescence quantum yield. It was shown that the main mechanism for the deactivation of the excited electronic energy of the first singlet excited state is the process of internal conversion.

Structural and functional analysis of HtrA1 and its subdomains.

The homotrimeric human serine protease HtrA1 is homologous to bacterial HtrA proteases regarding the trypsin-like catalytic and PDZ domains but differs by the presence of an N-terminal domain with IGFBP and Kazal homology. The crystal structures and SAXS analysis presented herein reveal the rare tandem of IGFBP- and Kazal-like modules, a protease active site that adopts a competent conformation in the absence of substrate or inhibitor and a model for the intact protein in solution. Highly sensitive enzymatic assays and binding studies demonstrate that the N-terminal tandem has no apparent effect on protease activity, and in accordance with the structure-based predictions, neither the IGFBP- nor Kazal-like module retains the function of their prototype proteins. Our structures of the unliganded HtrA1 active site suggest two-state equilibrium and a "conformational selection" model, in which substrate binds to the active conformer.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Differences in amino acid residues in the binding pockets dictate substrate specificities of mouse senescence marker protein-30, human paraoxonase1, and squid diisopropylfluorophosphatase.

Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed ß-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 was d-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The K(m) values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal-ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.

Hydrolysis potential of recombinant human skin and kidney prolidase against diisopropylfluorophosphate and sarin by in vitro analysis.

Human prolidase (PROL), which has structural homology to bacterial organophosphate acid anhydrolase that hydrolyze organophosphates and nerve agents has been proposed recently as a potential catalytic bioscavenger. To develop PROL as a catalytic bioscavenger, we evaluated the in vitro hydrolysis efficiency of purified recombinant human PROL against organophosphates and nerve agents. Human liver PROL was purified by chromatographic procedures, whereas recombinant human skin and kidney PROL was expressed in Trichoplusia ni larvae, affinity purified and analyzed by gel electrophoresis. The catalytic efficiency of PROL against diisopropylfluorophosphate (DFP) and nerve agents was evaluated by acetylcholinesterase back-titration assay. Partially purified human liver PROL hydrolyzed DFP and various nerve agents, which was abolished by specific PROL inhibitor showing the specificity of hydrolysis. Both the recombinant human skin and kidney PROL expressed in T. ni larvae showed ∼99% purity and efficiently hydrolyzed DFP and sarin. In contrast to human liver PROL, both skin and kidney PROL showed significantly low hydrolyzing potential against nerve agents soman, tabun and VX. In conclusion, compared to human liver PROL, recombinant human skin and kidney PROL hydrolyze only DFP and sarin showing the substrate specificity of PROL from various tissue sources.

Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate.

Rhomboid proteases have many important biological functions. Unlike soluble serine proteases such as chymotrypsin, the active site of rhomboid protease, which contains a Ser-His catalytic dyad, is submerged in the membrane and surrounded by membrane-spanning helices. Previous crystallographic analyses of GlpG, a bacterial rhomboid protease, and its complex with isocoumarin have provided insights into the mechanism of the membrane protease. Here, we studied the interaction of GlpG with 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate, both mechanism-based inhibitors for the serine protease, and describe the crystal structure of the covalent adduct between GlpG and diisopropyl fluorophosphonate, which mimics the oxyanion-containing tetrahedral intermediate of the hydrolytic reaction. The crystal structure confirms that the oxyanion is stabilized by the main chain amide of Ser-201 and by the side chains of His-150 and Asn-154. The phosphorylation of the catalytic Ser-201 weakens its interaction with His-254, causing the catalytic histidine to rotate away from the serine. The rotation of His-254 is accompanied by further rearrangement of the side chains of Tyr-205 and Trp-236 within the substrate-binding groove. The formation of the tetrahedral adduct is also accompanied by opening of the L5 cap and movement of transmembrane helix S5 toward S6 in a direction different from that predicted by the lateral gating model. Combining the new structural data with those on the isocoumarin complex sheds further light on the plasticity of the active site of rhomboid membrane protease.

Juvenile hormone (JH) esterase of the mosquito Culex quinquefasciatus is not a target of the JH analog insecticide methoprene.

Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (k(cat)/K(M) ratio) and V(max) values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE.

Structural insights into the conformational diversity of ClpP from Bacillus subtilis.

ClpP is a cylindrical protease that is tightly regulated by Clp-ATPases. The activation mechanism of ClpP using acyldepsipeptide antibiotics as mimics of natural activators showed enlargement of the axial entrance pore for easier processing of incoming substrates. However, the elimination of degradation products from inside the ClpP chamber remains unclear since there is no exit pore for releasing these products in all determined ClpP structures. Here we report a new crystal structure of ClpP from Bacillus subtilis, which shows a significantly compressed shape along the axial direction. A portion of the handle regions comprising the heptameric ring-ring contacts shows structural transition from an ordered to a disordered state, which triggers the large conformational change from an extended to an overall compressed structure. Along with this structural change, 14 side pores are generated for product release and the catalytic triad adopts an inactive orientation. We have also determined B. subtilis ClpP inhibited by diisopropylfluoro-phosphate and analyzed the active site in detail. Structural information pertaining to several different conformational steps such as those related to extended, ADEP-activated, DFP-inhibited and compressed forms of ClpP from B. subtilis is available. Structural comparisons suggest that functionally important regions in the ClpP-family such as N-terminal segments for the axial pore, catalytic triads, and handle domains for the product releasing pore exhibit intrinsically dynamic and unique structural features. This study provides valuable insights for understanding the enigmatic cylindrical degradation machinery of ClpP as well as other related proteases such as HslV and the 20S proteasome.

Interactions of phosphororganic agents with water and components of polyelectrolyte membranes.

Interactions of nerve G-agents (sarin and soman) and their simulants DMMP (dimethyl methylphosphonate) and DIFP (diisopropyl fluorophosphate) with water and components of polyelectrolyte membranes are studied using ab initio calculations in conjunction with thermodynamic modeling using the conductor-like screening model for real solvents (COSMO-RS). To test reliability of COSMO-RS calculations, we measured the vapor-liquid equilibrium in DMMP-water mixtures and found quantitative agreement between computed and experimental results. Using COSMO-RS, we studied the interactions of phosphororganic agents with the characteristic fragments of perfluorinated and sulfonated polystyrene (sPS) polyelectrolytes, which are explored for protective clothing membranes. We found that both simulants, DIFP and DMMP, mimic the thermodynamic properties of G-agents reasonably well; however, there are certain specific differences that are discussed. We also suggested that sPS-based polyelectrolytes have less affinity for phosphorganic agents compared to prefluorinated polyelectrolytes similar to Nafion.