High-activity enzyme-polyurethane coatings

The synthesis of water-borne polyurethane coatings in the presence of diisopropylfluorophosphatase (DFPase, E.C. 3.8.2.1) enabled the irreversible attachment of the enzyme to the polymeric matrix. The distribution of immobilized DFPase as well as activity retention are homogeneous within the coating. The resulting enzyme-containing coating (ECC) film hydrolyzes diisopropylfluorophosphate (DFP) in buffered media at high rates, retaining approximately 39% intrinsic activity. Decreasing ECC hydrophilicity, via the use of a less hydrophilic polyisocyanate during polymerization, significantly enhanced the intrinsic activity of the ECC. DFPase-ECC has biphasic deactivation kinetics, where the initial rapid deactivation of DFPase-ECC leads to the formation of a hyperstable and active form of enzyme.     

A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange

Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30–40 kDa to overcome problems arising from rapid ¹H and ¹³C transverse relaxation. However, ¹H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D₂O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H₂O. As a side-effect partial C protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary ¹H^N- and ¹H-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D₂O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of C protonation of individual amino acids is determined quantitatively and transverse relaxation properties of ¹H^N and ¹⁵N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.     

Simultaneous measurement of protein one-bond and two-bond nitrogen-carbon coupling constants using an internally referenced quantitative J-correlated [(15)N,(1)H]-TROSY-HNC experiment

A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone ¹ J _NC, ¹ J _NC, and ² J _NC coupling constants are obtained for the two [¹⁵N;¹³C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [²H;¹⁵N;¹³C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J _NC values on protein backbone torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated ¹⁵N-¹³C residual dipolar couplings.     

The first crystal structure of gluconolactonase important in the glucose secondary metabolic pathways

The first gluconolactonase crystal structure from bacteria has been determined to a resolution of 1.61 Å using X-ray crystallography. It belongs to the senescence marker protein 30/gluconolaconase superfamily but exhibits substrate specificity mainly toward d-glucono-δ-lactone. It forms a novel disulfide-bonded clamshell dimer comprising two doughnut-shaped six-bladed β-propeller domains, yet with an exceptionally long N-terminal subdomain forming an extra helix and four additional β-strands to enclose half of the outermost β-strands of each propeller. Extensive interactions, including H-bonds, salt bridges, disulfide bonds, and coordination bonds, along with numerous bridging water molecules, are present in the interface to institute the “top-to-top” clamshell-type dimer. Three calcium ions per subunit were observed. Two are present in the central water-filled channel, with the top one coordinated to four highly conserved amino acids and is possibly involved in substrate hydrolysis, while the bottom one is coordinated to the backbone oxygen atoms, which is possibly for stabilizing the propeller domain. One calcium ion is situated in the interface also to stabilize the dimer form. Since gluconolactonase is essential in the glucose secondary metabolic pathways leading to the synthesis of pentose, vitamin C, or “antiaging” factors, determination of its tertiary structure should help understand these important biochemical processes.     

Quantification of hydrolysis of toxic organophosphates and organophosphonates by diisopropyl fluorophosphatase from Loligo vulgaris by in situ Fourier transform infrared spectroscopy

The enzyme diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris effectively catalyzes the hydrolysis of diisopropyl fluorophosphate (DFP) and a number of organophosphorus nerve agents, including sarin, soman, cyclosarin, and tabun. Up to now, the determination of kinetic data has been achieved by techniques such as pH-stat titration, ion-selective electrodes, and fluorogenic substrate analogs. We report a new assaying method using in situ Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR) for the real-time determination of reaction rates. The method employs changes in the P-O-R stretching vibration of DFP and nerve agent substrates when hydrolyzed to their corresponding phosphoric and phosphonic acids. It is shown that the Lambert-Beer law holds and that changes in absorbance can be directly related to changes in concentration. Compared with other methods, the use of in situ FTIR spectroscopy results in a substantially reduced reaction volume that adds extra work safety when handling highly toxic substrates. In addition, the new method allows the noninvasive measurement of buffered solutions with varying ionic strengths complementing existing methods. Because the assay is independent of the used enzyme, it should also be applicable to other phosphotriesterase enzymes such as organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA), and paraoxonase (PON).     

猪肝DFPase的结构及某些性质的研究

 本文对纯化了的猪肝DFPase~[**]进行了一系列结构和性质的测定。用重复中毒的方法证明了猪肝中低分子量二异丙基氟磷酸酯(DFP)水解酶(DFPase)是属于催化酶。分析了DFPase的氨基酸组成并求出其分子量为17900。该酶的km值为6.7×10~(-4)mol/L。催化水解DFP的最适pH为7.5～7.7,反应最佳温度为70℃。根据催化水解DFP、Soman的活力以及Mn~(2+)对此酶的影响证明该酶属于Mazur type DFPase~[1]。     

Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

Methods are described to correlate aromatic ¹H ²/¹³C ² or ¹H ¹/¹⁵N ¹ with aliphatic ¹³C chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [¹⁵N, ¹H]- and/or [¹³C, ¹H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with -carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [¹⁵N, ¹H]-TROSY-H(N)C^ar experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine -CH and tryptophan -NH groups.