Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid.

We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a conventional molecular beacon. It was found that the overall sensitivities for the four PCR probes are in the order of MB>ATssMB>GCssMB>TaqMan. The fluorescence quantum yield measurements indicate that incomplete or partial enzymatic cleavage catalyzed by Taq polymerase is the likely cause of the low sensitivities of two shared-stem beacons when compared with the conventional beacon probe. A high-fluorescence background associated with the current TaqMan probe sequence contributes to the relatively low detection sensitivity and signal-to-background ratio. The study points out that the nucleotide environment surrounding the reporting fluorophore can strongly affect the probe performance in real-time PCR.

Spectroscopic characterization of fluorescein- and tetramethylrhodamine-labeled oligonucleotides and their complexes with a DNA template.

We measured absorption and emission spectra, fluorescence quantum yield, anisotropy, fluorescence resonance energy transfer (FRET), and melting temperature to characterize fluorescein- and tetramethylrhodamine (TMR)-labeled oligonucleotides in solution and when hybridized to a common DNA template. Upon hybridization to the template, both the absorption and emission spectra of TMR-labeled duplexes exhibited a shift with respect to those of labeled oligonucleotides, depending on the location of the TMR on the oligonucleotide. Measurements of quantum yield, anisotropy, and melting temperature indicated that TMR interacted with nucleotides within the duplexes in the order (T1>T5>T11, T16) that the oligonucleotide with TMR labeled at the 5' end (T1) is stronger than that labeled at position 5 from the 5' end (T5), which is also stronger than those labeled at the positions, 11 and 16, from the 5' end (T11, T16). In the case of the duplex formed between T1 and the template, fluorescence quenching was observed, which is attributed to the interaction between the dye molecule and guanosines located at the single-stranded portion of the template. A two-state model was suggested to describe the conformational states of TMR in the duplex. The melting temperatures of the four FRET complexes show the same pattern as those of TMR-labeled duplexes. We infer that the interactions between TMR and guanosine persist in the FRET complexes. This interaction may bring the donor and the acceptor molecules closely together, which could cause interaction between the two dye molecules shown in absorbance measurements of the FRET complexes.

Infantile hereditary spastic paraparesis due to codominant mutations in the spastin gene.

The authors describe an infant with a severe spastic paraparesis caused by two codominant mutations of the spastin gene. This highlights the multiple molecular mechanisms that are likely to be involved in the molecular pathology of SPG4 and illustrates the importance of complete screening of the spastin gene in affected individuals, particularly if the index case has an unusual phenotype.

Structure of alanine dehydrogenase from Archaeoglobus: active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin.

The hyperthermophilic archaeon Archaeoglobus fulgidus contains an L-Ala dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent dehydrogenases. The gene (Genbank; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.

Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template.

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.

Chorismate lyase: kinetics and engineering for stability.

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

The crystal structure of chorismate lyase shows a new fold and a tightly retained product.

The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4-hydroxy benzoate (4HB) for the ubiquinone pathway. In Escherichia coli, CL is monomeric, with 164 residues. We have determined the structure of the CL product complex by crystallographic heavy-atom methods and report the structure at 1.4-A resolution for a fully active double Cys-to-Ser mutant and at 2.0-A resolution for the wild-type. The fold involves a 6-stranded antiparallel beta-sheet with no spanning helices and novel connectivity. The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main-chain amides and by the buried side-chains of Arg 76 and Glu 155. The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix-turn-helix loops. The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10-fold stronger binding of product than substrate. Substrate binding and kinetically rate-limiting product release apparently require the rearrangement of these active-site-covering loops. Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane-bound enzyme 4HB octaprenyltransferase.

Improving the cytochrome P450 enzyme system for electrode-driven biocatalysis of styrene epoxidation.

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large-scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min(-1) by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, alpha-hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The alpha-hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.

Temperature dependence of the formal reduction potential of putidaredoxin.

Putidaredoxin (Pdx), a [2Fe-2S] redox protein of size M(r) 11,600, transfers two electrons in two separate steps from the flavin containing putidaredoxin reductase to the heme protein, cytochrome CYP101 in the P450cam catalytic cycle. It has recently come to light, through NMR measurements, that there can be appreciable differences in the Pdx conformational dynamics between its reduced and oxidized states. The redox reaction entropy, deltaS(0')rc = (S(0')Pdx(r)-S(0')Pdx(0)), as determined from measurements of the variation in formal potential with temperature, E0'(T), provides a measure of the strength of this influence on Pdx function. We designed a spectroelectrochemical cell using optically transparent tin oxide electrodes, without fixed or diffusible mediators, to measure E0'(T) over the temperature range 0-40 degrees C. The results indicate that the redox reaction entropy for Pdx is biphasic, decreasing from -213 +/- 27 J mol(-1) K(-1) over 0-27 degrees C, to -582 +/- 150 J mol(-1) K (-1) over 27-40 degrees C. These redox reaction entropy changes are significantly more negative than the changes reported for most cytochromes, although our measurement over the temperature interval 0-27 degrees C is in the range reported for other iron-sulfur proteins. This suggests that Pdx (and other ferredoxins) is a less rigid system than monohemes, and that redox-linked changes in conformation, and/or conformational dynamics, impart to these proteins the ability to interact with a number of redox partners.

Thermodynamics of reactions catalyzed by anthranilate synthase.

Microcalorimetry and high performance liquid chromatography have been used to conduct a thermodynamic investigation of reactions catalyzed by anthranilate synthase, the enzyme located at the first step in the biosynthetic pathway leading from chorismate to tryptophan. One of the overall biochemical reactions catalyzed by anthranilate synthase is: chorismate(aq) + ammonia(aq) = anthranilate(aq) + pyruvate(aq) + H2O(l). This reaction can be divided into two partial reactions involving the intermediate 2-amino-4-deoxyisochorismate (ADIC): chorismate(aq) + ammonia(aq) = ADIC(aq) + H2O(l) and ADIC(aq) = anthranilate(aq) + pyruvate(aq). The native anthranilate synthase and a mutant form of it that is deficient in ADIC lyase activity but has ADIC synthase activity were used to study the overall ammonia-dependent reaction and the first of the above two partial reactions, respectively. Microcalorimetric measurements were performed on the overall reaction at a temperature of 298.15 K and pH 7.79. Equilibrium measurements were performed on the first partial (ADIC synthase) reaction at temperatures ranging from 288.15 to 302.65 K, and at pH values from 7.76 to 8.08. The results of the equilibrium and calorimetric measurements were analyzed in terms of a chemical equilibrium model that accounts for the multiplicity of ionic states of the reactants and products. These calculations gave thermodynamic quantities at the temperature 298.15 K and an ionic strength of zero for chemical reference reactions involving specific ionic forms. For the reaction: chorismate2-(aq) + NH4+(aq) = anthranilate-(aq) + pyruvate-(aq) + H+(aq) + H2O(l), delta rHmo = -(116.3 +/- 5.4) kJ mol-1. For the reaction: chorismate2-(aq) + NH4+(aq) = ADIC-(aq) + H2O(l), K = (20.3 +/- 4.5) and delta rHmo = (7.5 +/- 0.6) kJ mol-1. Thermodynamic cycle calculations were used to calculate thermodynamic quantities for three additional reactions that are pertinent to this branch point of the chorismate pathway. The quantities obtained in this study permit the calculation of the position of equilibrium of these reactions as a function of temperature, pH, and ionic strength. Values of the apparent equilibrium constants and the standard transformed Gibbs energy changes delta rG'mo under approximately physiological conditions are given.

Crystallization and 1.1-A diffraction of chorismate lyase from Escherichia coli.

Chorismate pathway enzymes are important as producers of nonnucleotide aromatic compounds. The enzyme chorismate lyase from Escherichia coli has been crystallized in four distinct forms, three of which have been characterized by X-ray diffraction. Despite widespread screening, all four crystal forms grow from the same chemical conditions. The wild-type enzyme tends to aggregate, even in the presence of reducing agent, and yielded only one crystal form (monoclinic, form 1) that grew in intricate clusters. Chemical modification of the cysteines mitigated problems with aggregation and solubility but did not affect crystal growth behavior. Protein aggregation was largely eliminated by mutating the protein's two cysteines to serines. The double mutant retains full enzymatic activity and crystallizes in three new forms, one of which (triclinic) diffracts to 1.1-A resolution.

Comparison of backbone dynamics of oxidized and reduced putidaredoxin by 15N NMR relaxation measurements.

The backbone dynamics of uniformly 15N-labeled reduced and oxidized putidaredoxin (Pdx) have been studied by 2D 15N NMR relaxation measurements. 15N T1 and T2 values and 1H-15N NOEs have been measured for the diamagnetic region of the protein. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameters (S2), the effective correlation time for internal motions (tau e), and the 15N exchange broadening contributions (Rex) for each residue, as well as the overall correlation time (tau(m)). Order parameters for the reduced Pdx are generally higher than for the oxidized Pdx, and there is increased mobility on the microsecond to millisecond time scale for the oxidized Pdx, in comparison with the reduced Pdx. These results clearly indicate that the oxidized protein exhibits higher mobility than the reduced one, which is in agreement with the recently published redox-dependent dynamics studied by amide proton exchange. In addition, we observed very high T1/T2 ratios for residues 33 and 34, giving rise to a large Rex contribution. Residue 34 is believed to be involved in the binding of Pdx to cytochrome P450cam (CYP101). The differences in the backbone dynamics are discussed in relation to the oxidation states of Pdx, and their impact on electron transfer. The entropy change occurring on oxidation of reduced Pdx has been calculated from the order parameters of the two forms.

Exploration of the structural environment of the iron-sulfur cluster in putidaredoxin by nitrogen-15 NMR spectroscopy of selectively labeled cysteine residues.

Putidaredoxin is a di-iron protein whose paramagnetic region is not well characterized by 1H detected NMR. We have studied the structure of this region in greater detail by directly observed 15N NMR of oxidized and reduced putidaredoxin preparations in which the six cysteine residues are selectively labeled with 15N. A new method for preparation of a stable form of reduced putidaredoxin has been developed for use in NMR. The 15N NMR spectra of the oxidized and reduced forms are characteristically different, and we have measured and compared 15N chemical shifts, spin-lattice relaxation times (T1), and chemical shift/temperature dependences for both forms. Evidence for localized valencies of the iron atoms in the reduced form is presented. From the 15N T1 values of the oxidized form, reduced distances of the cysteine backbone 15N nuclei from the center of the Fe2S2 cluster have been calculated. These distances are consistent with those calculated from X-ray crystal structure data for five ferredoxins, and confirm the structural similarity of the Fe2S2 clusters in putidaredoxin and in these ferredoxins in the oxidized state.

Cu2+ Reduction by Tomato Root Plasma Membrane Vesicles.

Reduction of Cu2+ by plasma membrane vesicles isolated from tomato (Lycopersicon esculentum Mill.) roots was investigated. Plants were grown in hydroponic culture with complete nutrition for 4 weeks or were deprived of Fe for the last 7 d. Plasma membrane vesicles were prepared by aqueous two-phase partitioning. Reduction of Cu, Fe, and ferricyanide by plasma membrane vesicles was measured. An increase in the activity of all three pyridine-nucleotide-dependent activities was noted in plasma membrane preparations from Fe-deficient, compared to Fe-sufficient, plants. Solubilization and chromatographic separation of two plasma membrane electron transport systems indicated that the Fe-chelate reductase was probably responsible for reduction of Cu. Assays used a variety of Cu chelates, and for each the Cu activity in the assay was determined by the program Geochem PC. The rate of reduction of Cu correlated with the level of Cu activity, and results support the idea that free Cu2+ and not Cu chelates may serve as the true substrate for reduction. Reduction was observed only in assays in which Cu activity was equivalent to Cu-enriched or Cu-toxic soils. These results suggest that reduction of Cu by tomato root may have little or no physiological relevance under conditions experienced by the root in the soil.

The outer mitochondrial membrane channel, VDAC, is modulated by a protein localized in the intermembrane space.

The mitochondrial outer membrane channel, VDAC, provides a pathway for the flux of metabolites between the cytoplasm and mitochondrion. VDAC is voltage-dependent and occupies states of differing conductivity and ion selectivity that are dependent on transmembrane potential. A protein, derived from preparations of mitochondria, has been shown to increase the voltage dependence of VDAC and is called the VDAC modulator. Both VDAC and the VDAC modulator have been extensively characterized by reconstitution into planar lipid bilayers. In order for the VDAC modulator to have physiological significance it must have physical access to VDAC in the cell. This constraint dictates that the modulator be an extrinsic outer mitochondrial membrane protein, occupy the mitochondrial intermembrane space, or be a cytoplasmic constituent. To address the question of subcellular localization, purified mitochondria were selectively lysed with digitonin or treated with trypsin while resuspended in hypo-osmotic or iso-osmotic medium. Marker enzymes and modulator activity were monitored during the various treatments. Results indicate that the integrity of the outer membrane was necessary to prevent modulator release or protection from trypsin digestion. Outer membrane lysis, under conditions where the inner membrane remained intact, resulted in modulator release or inactivation by trypsin. These results suggest an intermembrane space location for the VDAC modulator in the mitochondrion.

Fe-Chelate Reductase Activity of Plasma Membranes Isolated from Tomato (Lycopersicon esculentum Mill.) Roots : Comparison of Enzymes from Fe-Deficient and Fe-Sufficient Roots.

Reduction of Fe(3+) to Fe(2+) is a prerequisite for Fe uptake by tomato roots. Ferric chelate reductase activity in plasma membranes (PM) isolated from roots of both iron-sufficient (+Fe) and iron-deficient (-Fe) tomatoes (Lycopersicon esculentum Mill.) was measured as NADH-dependent ferric citrate reductase and exhibited simple Michaelis-Menten kinetics for the substrates, NADH and Fe(3+)(citrate(3-))(2). NADH and Fe(3+)(citrate(3-))(2)K(m) values for reductase in PM from +Fe and -Fe tomato roots were similar, whereas V(max) values were two- to threefold higher for reductase from -Fe tomatoes. The pH optimum for Fe-chelate reductase was 6.5. Fe-chelate reductases from -Fe and +Fe tomato roots were equally sensitive to several triazine dyes. Reductase was solubilized with n-octyl beta-d-glucopyranoside and electrophoresed in nondenaturing isoelectric focusing gels. Three bands, with isoelectric points of 5.5 to 6.2, were resolved by enzyme activity staining of electrofocused PM proteins isolated from +Fe and -Fe tomato roots. Activity staining was particularly enhanced in the isoelectric point 5.5 and 6.2 bands solubilized from -Fe PM. We conclude that PM from roots of +Fe and -Fe plants contain Fe-chelate reductases with similar characteristics. The response to iron deficiency stress likely involves increased expression of constitutive Fe-chelate reductase isoforms in expanding epidermal root PM.

Effects of Helminthosporium maydis Race T Toxin on Electron Transport in Susceptible Corn Mitochondria and Prevention of Toxin Actions by Dicyclohexylcarbodiimide.

The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca(2+) gradients, induced by the toxin in T corn was prevented by N,N'-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N'-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site.

The mitochondrial outer membrane channel, VDAC, is modulated by a soluble protein.

The mitochondrial outer membrane channel, VDAC, serves as the primary permeability pathway for metabolite flux between cytoplasmic and mitochondrial compartments. VDAC can occupy several conformational states differing in ion conductivity. Small transmembrane potentials cause transitions from open- to closed-channel conformations. A soluble mitochondrial protein enhances the channel's response to voltage by increasing the rate of channel closing; inducing the occupation of lower conductance states; and decreasing the rate of channel reopening. This protein modulator acts at very low concentrations and its role in the cell may be to regulate the permeability of the mitochondrial outer membrane by inducing channel closure.

Dissipation of the Membrane Potential in Susceptible Corn Mitochondria by the Toxin of Helminthosporium maydis, Race T, and Toxin Analogs.

We have tested directly the effect of Helminthosporium maydis T (Hmt) toxin and various analogs on the membrane potential formed in mitochondria isolated from a Texas (T) cytoplasmic male-sterile and a normal (N) corn. ATP, malate or succinate generated a membrane potential (negative inside) as monitored by the absorbance change of a cationic dye, safranine. The relative membrane potential (Deltapsi) could also be detected indirectly as (45)Ca(2+) uptake. Hmt toxin added to T mitochondria dissipated the steady state Deltapsi similar to addition of a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Toxin analogs (Cpd XIII: C(41)H(68)O(12) and Cpd IV: C(25)H(44)O(6)), reduced native toxin (RT2C: C(41)H(84)O(13)) and Pm toxin (band A: C(33)H(60)O(8), produced by the fungus, Phyllosticta maydis) were effective in dissipating Deltapsi and decreasing Ca(2+) uptake with the following order: Pm (100) >> HmT (23-30) > Cpd XIII (11-25) >> RT2C (0-4-1.8) > Cpd IV (0.2-1.0). In contrast, the toxins and analogs had no effect on Deltapsi formed in N mitochondria. The striking similarities of the HmT toxin (band 1: C(41)H(68)O(13)) and Cpd XIII on T mitochondrial activities provide strong evidence supporting the correctness of the polyketol structure assigned to the native toxin. Since the Deltapsi in energized mitochondria is caused mainly by the electrogenic extrusion of H(+), the results support the idea that HmT toxin increases membrane permeability of T mitochondria to H(+). The host specificity of the toxin suggests that an interaction with unique target site(s) on the inner mitochondrial membrane of T corn causes H(+) leakage.