Improved purification and PCR amplification of DNA from environmental samples

Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA.     

<Superscript>13</Superscript>C-detected NMR experiments for measuring chemical shifts and coupling constants in nucleic acid bases

The paper presents a set of two-dimensional experiments that utilize direct ¹³C detection to provide proton–carbon, carbon–carbon and carbon–nitrogen correlations in the bases of nucleic acids. The set includes a ¹³C-detected proton–carbon correlation experiment for the measurement of ¹³C–¹³C couplings, the CaCb experiment for correlating two quaternary carbons, the HCaCb experiment for the ¹³C–¹³C correlations in cases where one of the carbons has a proton attached, the HCC-TOCSY experiment for correlating a proton with a network of coupled carbons, and a ¹³C-detected ¹³C–¹⁵N correlation experiment for detecting the nitrogen nuclei that cannot be detected via protons. The IPAP procedure is used for extracting the carbon–carbon couplings and/or carbon decoupling in the direct dimension, while the S³E procedure is preferred in the indirect dimension of the carbon–nitrogen experiment to obtain the value of the coupling constant. The experiments supply accurate values of ¹³C and ¹⁵N chemical shifts and carbon–carbon and carbon–nitrogen coupling constants. These values can help to reveal structural features of nucleic acids either directly or via induced changes when the sample is dissolved in oriented media. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

F nuclear magnetic resonance screening of glucokinase activators

Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R₂ relaxation of the ¹⁹F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the ¹⁹F signal intensity after an 80-ms spin echo correlates nicely with the EC₅₀, opening a route for NMR screening of GK activators.     

Newcastle disease virus (NDV) induces protein oxidation and nitration in brain and liver of chicken: Ameliorative effect of vitamin E

The present study was aimed at investigating the therapeutic efficacy of vitamin E on oxidative injury in brain and liver of Newcastle disease virus (NDV) challenged chickens. We have analyzed the xanthine oxidase (XOD) activity; uric acid (UA) levels and superoxide radical generation by using electron spin resonance spectroscopy. Further, protein oxidation, nitration and apoptosis were evaluated in the brain and liver of the control, NDV-infected and NDV + Vit. E treated groups. A significant elevation was observed in XOD activity and UA levels in brain (p < 0.001) and liver (p < 0.05) of NDV infected birds when compared to controls. Further, significant increase in the production of superoxides, enhanced intracellular protein carbonyls and nitrates were observed in the brain and liver of NDV-infected birds over healthy subjects. Apoptosis studies also suggested that a larger number of TUNEL positive cells were observed in brain and a moderately in liver of NDV-infected chickens. However, all these perturbations were significantly ameliorated in NDV + Vit. E treated chickens as compared to NDV-infected birds. Taken together, our results suggested that NDV-induced neuronal and hepatic damage at least in part mediates oxidative stress and on the other hand, supplementation of vitamin E mitigates NDV-induced oxidative damage thereby protects brain and liver of chickens. These findings could provide new insights into the understanding of NDV pathogenesis and therapeutic effects of dietary antioxidants.     

Structural insights into a low-spin myoglobin variant with bis-histidine coordination from molecular modeling

Rational design of functional enzymes is a powerful strategy to gain deep insights into more complex native enzymes, such as nitric oxide reductase (NOR). Recently, we engineered a functional model of NOR by creating a two His and one Glu (2‐His‐1‐Glu) non‐heme iron center in sperm whale myoglobin (swMb L29E, F43H, H64, called Fe_BMb(‐His)). It was found that Fe_BMb(‐His) adopts a low‐spin state with bis‐His coordination in the absence of metal ions binding to the designed metal center. However, no structural information was available for the variant in this special spin state. We herein performed molecular modeling of Fe_BMb(‐His) and compared with the X‐ray structure of its copper bound derivative, Cu(II)‐CN^?‐Fe_BMb(‐His), resolved recently at a high resolution (1.65 Å) (PDB entry 3MN0). The simulated structure shows that mutation of Leu to Glu at position 29 in the hydrophobic heme pocket alters the folding behavior of Mb. The hydrogen bond between Glu29 and His64 further plays a role in stabilizing the bis‐His (His64/His93) coordination structure. This study offers an excellent example of using molecular modeling to gain insights in rational design of both structural and functional proteins. Proteins 2011. © 2010 Wiley‐Liss, Inc.     

Characterization of the disordered-to-α-helical transition of IA₃ by SDSL-EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy coupled with site-directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA₃ is used as a model system demonstrating SDSL-EPR characterization of conformational changes in small IDP systems. IA₃ has 68 amino acids, is unstructured in solution, and becomes α-helical upon addition of the secondary structural stabilizer 2,2,2-trifluoroethanol (TFE). Two single cysteine substitutions, one in the N-terminus (S14C) and one in the C-terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h₍₊₁₎/h₍₀₎, the local tumbling volume (V_L), and the percent change of the h₍₋₁₎ intensity were utilized to quantitatively monitor TFE-induced conformational changes. The values of h_(+1)/h₍₀₎ as a function of TFE titration varied in a sigmoidal manner and were fit to a two-state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA₃. The other parameters provide site-specific information and show that S14C-SL undergoes a conformational change resulting in more restricted motion than N58C-SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α-helical propensity of the N-terminal segment of IA₃. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured-to-helical conformations in IDPs by SDSL.     

Recombinant phycobiliproteins. Recombinant C-phycocyanins equipped with affinity tags, oligomerization, and biospecific recognition domains

A family of specific cloning vectors was constructed to express in the cyanobacterium Anabaena sp. PCC7120 recombinant C-phycocyanin subunits with one or more different tags, including the 6xHis tag, oligomerization domains, and the streptavidin-binding Strep2 tag. Such tagged alpha or beta subunits of Anabaena sp. PCC7120 C-phycocyanin formed stoichiometric complexes in vivo with appropriate wild-type subunits to give constructs with the appropriate oligomerization state and normal posttranslational modifications and with spectroscopic properties very similar to those of unmodified phycocyanin. All of these constructs were incorporated in vivo into the rod substructures of the light-harvesting complex, the phycobilisome. The C-terminal 114-residue portion of the Anabaena sp. PCC7120 biotin carboxyl carrier protein (BCCP114) was cloned and overexpressed and was biotinylated up to 20% in Escherichia coli and 40% in wild-type Anabaena sp. His-tagged phycocyanin beta--BCCP114 constructs expressed in Anabaena sp. were >30% biotinylated. In such recombinant phycocyanins equipped with stable trimerization domains, >75% of the fusion protein was specifically bound to streptavidin- or avidin-coated beads. Thus, the methods described here achieve in vivo production of stable oligomeric phycobiliprotein constructs equipped with affinity purification tags and biospecific recognition domains usable as fluorescent labels without further chemical manipulation.     

ESEEM and Mössbauer studies of the ferriheme model compound bis(3-aminopyrazole)tetraphenylporphyrinatoiron(III) chloride, [TPPFe(NH2PzH)2]Cl

A model heme complex, bis(3-aminopyrazole)tetraphenylporphinatoiron(III) chloride, [TPPFe (NH₂PzH)₂]Cl, for which the EPR g-values lead to a rhombicity V/Δ=1.2 if g _zz is the largest g-value, have been investigated by electron spin echo envelope modulation (ESEEM) and Mössbauer spectroscopies. The ESEEM studies focus on the proton sum frequency peaks at near twice the proton Larmor frequency. Analysis of the distant proton peak (mainly due to the pyrrole-H) at exactly twice the proton Larmor frequency shows conclusively that g _zz is aligned along the normal to the porphyrin plane, and thus the electron configuration is (d _xy )²(d _xz ,d _yz )³, with g _zz >g _yy >g _xx . This system is thus another violation to Taylor's "proper axis system" rule. The near proton (the α-H and N-H of the axial ligands) peaks provide distance information for those protons from the metal. Magnetic Mössbauer studies of the same complex confirm the (d _xy )²(d _xz ,d _yz )³ ground state and indicate that, as is the case for cytochrome P450_cam, A _xx is the largest magnitude A-value, and is negative in sign. Other low-spin iron(III) porphyrinates also have A _xx of negative sign, but usually the magnitude is only about half that of A _zz , which is always positive in sign.     

A New Amide Proton R 1ρ Experiment Permits Accurate Characterization of Microsecond Time-scale Conformational Exchange

A new off-resonance spin-lock experiment to record relaxation dispersion profiles of amide protons is presented. The sensitivity-enhanced HSQC-type sequence is designed to minimize the interference from cross-relaxation effects and ensure that the dispersion profiles in the absence of μs-ms time-scale dynamics are flat. Toward this end (i) the proton background is eliminated by sample deuteration (Ishima et al., 1998), (ii) ¹H spin lock is applied to two-spin modes 2(H_xSin ϑ + H_zCos ϑ) N_z, and (iii) the tilt angle ϑ ≈ 35° is maintained throughout the series of measurements (Desvaux et al. Mol. Phys., 86 (1995) 1059). The relaxation dispersion profiles recorded in this manner sample a wide range of effective rf field strengths (up to and in excess of 20 kHz) which makes them particularly suitable for studies of motions on the time scale ≤100 μs. The new experiment has been tested on the Ca²⁺-loaded regulatory domain of cardiac troponin C. Many residues show pronounced dispersions with remarkably similar correlation times of 30 μs. Furthermore, these residues are localized in the regions that have been previously implicated in conformational changes (Spyracopoulos et al. Biochemistry, 36 (1997) 12138) Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.