Biochemical characterization of RNase HII from Aeropyrum pernix

Aeropyrum pernix contains one homolog of ribonuclease H (RNase H), A. pernix RNase HII (Ape-RNase HII). Activity characterization showed that Ape-RNase HII exhibited the highest activity in the presence of 5 mM Mn(2+), 1 mM Co(2+), or 10 mM Mg(2+), respectively; however, its cleavage efficiencies at different cleavage sites for Mn(2+) and Mg(2+) were different. Ape-RNase HII cleaved 12-bp RNA/DNA substrates at multiple sites and the optimum pH value was 11.0. Moreover, 16-bp DNA-r4-DNA/DNA and 13-bp DNA-r1-DNA/DNA chimeric substrates were cleaved at DNA-RNA junction. Ape-RNase HII was thermostable and the stabilization was enhanced with increased salt concentration. This work is believed to be the first in vitro functional study of Ape-RNase HII and the results should contribute to the analysis of RNase H of other archaeal species.     

Provirus induction in hyperthermophilic archaea: characterization of Aeropyrum pernix spindle-shaped virus 1 and Aeropyrum pernix ovoid virus 1

By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum pernix, and under special conditions of A. pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae.     

Biochemical characterization of DNA-binding proteins from Pyrobaculum aerophilum and Aeropyrum pernix

Several representatives of the Crenarchaeal branch of the Archaea contain highly abundant, small, positively charged proteins exemplified by the Sso7d protein from Sulfolobus solfataricus. These proteins bind to DNA in a non-sequence-specific manner. Using publicly available genomic sequence information, we identified a second class of small Crenarchaeal DNA-binding proteins represented by the Pyrobaculum aerophilum open reading frame 3192–encoded (Pae3192) protein and its paralogs. We investigated the biochemical properties of the Pae3192 protein and an orthologous protein (Ape1322b) from Aeropyrum pernix in side-by-side experiments with the Sso7d protein. We demonstrate that the recombinant Ape1322b, Pae3192 and Sso7d proteins bind to DNA and that the DNA-protein complexes formed are slightly different for each protein. We show that like Sso7d, Pae3192 constrains negative supercoils in DNA. In addition, we show that all three proteins raise the melting temperature of duplex DNA upon binding. Finally, we present the equilibrium affinity constants and kinetic association constants of each protein for single-stranded and double-stranded DNA.     

Study of the thermal behavior of azidohetarenes with differential scanning calorimetry

Thermal properties of azidohetarenes without reactive ortho-substituents, obtained by DSC analysis, were compared with the DSC data of ortho-phenyl and ortho-acyl substituted azidohetarenes, which give ring closure reactions either to indoles or to five-membered heterocycles such as isoxazoles. The reaction temperatures and reaction enthalpies give both information to prepare the reaction conditions and important safety information and were, in addition, used to find out relations between the temperatures or enthalpies with the three different reaction mechanisms.     

Determination of hydration properties and thermal behavior of Paecilomyces variotii by differential scanning calorimetry

Due to the structure and the composition of Paecilomyces variotii, the mycelia of this fungus could have potential applications as ingredients in wettable foods. For this use, drying could be employed, justifying the study of thermal behavior of P. variotii. The objectives of this work were to perform a study of thermal behavior of P. variotii isolates, to evaluate the hydration properties of these mycelia and to analyze the effect of different technological parameters on the latter properties. Wet cultures exhibited a wide endothermic transition, with mean values of peak temperature of 61°C and denaturation enthalpy of 4 J/g dry matter. Initial (50°C) and final (80°C) temperatures of the endothermic transition were used to dry the mycelia. Freeze-drying was also assayed. For all dried mycelia, a decrease in denaturation enthalpy between 40 and 50% was observed for drying at 50°C and freeze-drying, and a drastic decrease of almost 100% for drying at 80°C. According to the hydration properties, wet mycelia exhibited water holding capacity (WHC) value of 45 g water/g dry matter. Significant differences among dried mycelia, resulting WHC values in order: 50°C > freeze-dried > 80°C (p < 0.05) were revealed for each P. variotii strain. Fungi obtained by drying at 50 C and by freeze-drying, showed a rapid water absorption (t _1/2 < 0.1 min). Ionic strength, pH and particle size of dried mycelia influenced the hydration properties.     

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 μM, a K(M) for phosphoenolpyruvate of 891 μM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.     

Comparison of the stabilities and unfolding pathways of human apolipoprotein E isoforms by differential scanning calorimetry and circular dichroism

Differential scanning calorimetry and circular dichroism experiments were performed to study structural differences among the common isoforms of human apolipoprotein E (apoE2, apoE3, and apoE4) and their N-terminal, 22-kDa fragments. Here, we examine thermodynamic properties that characterize the structural differences among isoforms, and also differences in their unfolding behavior. The 22-kDa fragments and their full-length counterparts were found to exhibit similar differences in thermal stability (apoE4相似文献   

Physical-chemical characterization and stability study of alpha-trypsin at pH 3.0 by differential scanning calorimetry

alpha-Trypsin is a serine-protease with a polypeptide chain of 223 amino acid residues and six disulfide bridges. It is a globular protein with predominance of antiparallel ss-sheet secondary structure and it has two domains with similar structures. In the present work, a stability study of alpha-trypsin in the acid pH range was performed and some physical-chemical denaturation parameters were measured by using differential scanning calorimetry (DSC). The alpha-trypsin has a shelf-life (t(95%)) of about 10 months at pH 3.0 and 4 degrees C and its hydrolysis into the psi-trypsin isoform is negligible during 6 months. The observed ratio DeltaH(cal)/DeltaH(vH) is close to unity, which suggests the occurrence of a two-state transition. At pH 3.0, alpha-trypsin unfolded with T(m) = 325.9 K and DeltaH = 99.10 kcal mol(-1), and the change in heat capacity between the native and unfolded forms of the protein was estimated to be 1.96+/-0.18 kcal mol(-1)K(-1). The stability of alpha-trypsin calculated at 298 K was DeltaG(U)=6.10 kcal mol(-1) at pH 3.0. These values are in the range expected for a small globular protein. These results show that the thermodynamic parameters of unfolding of beta-trypsin do not change substantially after its conversion to alpha-trypsin.     

Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1.

We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrum pernix K1. The gene (Accession no. APE0641) of A. pernix encoding a 37 kDa protein contains a redox active site motif (CPHC) but its N-terminal extension region (about 200 residues) shows no homology within the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH-dependent protein disulfide reductase system in the insulin reduction. In addition, the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 degrees C, and the half-life of heat inactivation was about 4 min at 110 degrees C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about -262 mV, which was slightly higher than that of Trx from E. coli (-270 mV). These results indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria.     

Thermal denaturation of whole cells and cell components of Escherichia coli examined by differential scanning calorimetry.

Thermograms of whole cells of Escherichia coli obtained by differential scanning calorimetry contained ten main peaks (denoted f, l, m1, m2, m3, n, p, q, r and s) occurring at temperatures of approximately 25, 54, 61, 71, 76, 81, 95, 105, 118 and 124 degrees C, respectively. After cooling to 5 degrees C and reheating, peaks denoted fr, mr and pr were observed at 23, 73 and 94 degrees C, respectively. By examining thermograms of different cell fractions we have identified the following thermal denaturation events. During primary heating there is a broad endotherm (f) beginning below 20 degrees C and extending to just above 40 degrees C that is caused by melting of membrane lipids. Superimposed on this is an exothermic process associated with a change of state of the peptidoglycan. The first irreversible denaturation event occurs just above 47 degrees C, associated with the onset of denaturation of the 30S ribosomal subunit and soluble cytoplasmic proteins. Ribosome melting is a complex process occurring between 47 and 85 degrees C and is characterized by peaks m1, m2 and n. Peak m3 at 75-76 degrees C is of unknown identity but may possibly represent melting of tRNA. Peak p at 95 degrees C results from melting of a portion of the cellular DNA combined with denaturation of a cell wall component. Peak q at 105 degrees C is multicomponent and may be caused by melting of a different region of DNA together with denaturation of another cell wall component. The complex events denoted r and s at 118 and 125 degrees C, respectively, are associated with denaturation of a component of the cell envelope, and possibly also of DNA. Following cooling and reheating there is a broad endotherm with a maximum at 23 degrees C caused by remelting of membrane lipid and a very broad endotherm extending between 40 and 100 degrees C caused by the remelting of ribosomal RNA. Peak pr at 94 degrees C is caused by the melting of reannealed DNA. Additional features not appearing in whole cells were evident in some cell fractions. These observations should allow us to distinguish events that may lead to loss of viability from those that do not.     

Dynamic analysis of differential scanning calorimetry data

The apparent heat capacity function measured by high-sensitivity differential scanning calorimetry contains dynamic components of two different origins: (1) an intrinsic component arising from the finite instrument time response; and (2) a sample component arising from the kinetics of the thermal transition under study. The intrinsic instrumental component is always present and its effect on the shape of the experimental curve depends on the magnitude of the calorimeter response time. Usually, high-sensitivity instruments exhibit characteristic time constants varying from 10 to 100 s. This slow response introduces distortions in the shape of the heat capacity function especially at fast scanning rates. In addition to this instrumental component, dynamic effects due to sample relaxation processes also contribute to the shape of the experimental heat capacity profile. Since the nature and magnitude of these effects are a function of the kinetic parameters of the transition, they can be used to obtain kinetic information. This communication presents a dynamic deconvolution technique directed to remove artificial distortions in the shape of the heat capacity function measured at any scanning rate, and to obtain a kinetic characterization of a thermally induced transition. The kinetic characterization obtained by this method allows the researcher to obtain transition relaxation times as a continuous function of temperature. This technique has been applied to the thermal unfolding of ribonuclease A and the pretransition of dipalmitoylphosphatidylcholine (DPPC). In both systems the transition relaxation times are temperature dependent. For the protein system the relaxation time is very slow below the transition temperature (approximately 30 s) and very fast above Tm (less than 1 s) in agreement with direct kinetic measurements. For the pretransition of DPPC, the relaxation time is maximal at the transition midpoint and of the order of approx. 40 s.     

Thermal inactivation of Listeria monocytogenes studied by differential scanning calorimetry

The effect of NaCl on the thermal inactivation of Listeria monocytogenes has been investigated by conventional microbiological techniques and by using differential scanning calorimetry (DSC). Addition of 1.5 M-NaCl to cells grown at lower NaCl concentrations significantly increases the tolerance of cells to mild heat stress (56-62 degrees C). DSC thermograms show five main peaks which are shifted to higher temperatures in the presence of 1.5 M-NaCl. Measurement of loss of viability in the calorimeter gave good correlation between cell death and the first major thermogram peak at two NaCl concentrations. The time course of the loss of this first peak when cells were heated and held at 60 degrees C in the calorimeter matched the loss of viability, whereas the peak attributable to DNA showed little change during this process. The use of DSC to investigate the mechanisms involved in thermal inactivation is discussed.     

Statistical analysis of plasma thermograms measured by differential scanning calorimetry

Melting curves of human plasma measured by differential scanning calorimetry (DSC), known as thermograms, have the potential to markedly impact diagnosis of human diseases. A general statistical methodology is developed to analyze and classify DSC thermograms to analyze and classify thermograms. Analysis of an acquired thermogram involves comparison with a database of empirical reference thermograms from clinically characterized diseases. Two parameters, a distance metric, P, and correlation coefficient, r, are combined to produce a 'similarity metric,' ρ, which can be used to classify unknown thermograms into pre-characterized categories. Simulated thermograms known to lie within or fall outside of the 90% quantile range around a median reference are also analyzed. Results verify the utility of the methods and establish the apparent dynamic range of the metric ρ. Methods are then applied to data obtained from a collection of plasma samples from patients clinically diagnosed with SLE (lupus). High correspondence is found between curve shapes and values of the metric ρ. In a final application, an elementary classification rule is implemented to successfully analyze and classify unlabeled thermograms. These methods constitute a set of powerful yet easy to implement tools for quantitative classification, analysis and interpretation of DSC plasma melting curves.     

Analysis of the thermal unfolding of porcine procarboxypeptidase A and its functional pieces by differential scanning calorimetry

Porcine pancreatic procarboxypeptidase A and its tryptic peptides, carboxypeptidase A and the activation segment, have been studied by high-sensitivity differential scanning calorimetry (DSC). The thermal denaturation of the zymogen and the active enzyme has been carried out at two pH values, 7.5 and 9.0, at different ionic strengths and at different scan rates. The endothermic transitions for these two proteins were always irreversible under all conditions investigated. The denaturation behaviour of both proteins seems to fit very well with the kinetic model for the DSC study of irreversible unfolding of proteins recently proposed by one of our groups. From this model, the activation energies obtained for the denaturation of the pro- and carboxypeptidase were 300 +/- 20 kJ mol-1 and 250 +/- 14 kJ mol-1 respectively. On the other hand, the isolated activation segment appears as a thermostable piece with a highly reversible thermal unfolding which follows a two-state process. The denaturation temperature observed for the isolated segment was always at least 15 K higher than those of the zymogen and the active enzyme.     

Study of thermal properties and heat-induced denaturation and aggregation of soy proteins by modulated differential scanning calorimetry

The thermal properties and heat-induced denaturation and aggregation of soy protein isolates (SPI) were studied using modulated differential scanning calorimetry (MDSC). Reversible and non-reversible heat flow signals were separated from the total heat flow signals in the thermograms. In the non-reversible profiles, two major endothermic peaks (at around 100 and 220 degrees C, respectively) associated with the loss of residual water were identified. In the reversible profiles, an exothermic peak associated with thermal aggregation was observed. Soy proteins denatured to various extents by heat treatments showed different non-reversible and reversible heat flow patterns, especially the exothermic peak. The endothermic or exothermic transition characteristics in both non-reversible and reversible signals were affected by the thermal history of the samples. The enthalpy change of the exothermic (aggregation) peak increased almost linearly with increase in relative humidity (RH) in the range between 8 and 85%. In contrast, the onset temperature of the exotherm decreased progressively with increase in RH. These results suggest that the MDSC technique could be used to study thermal properties and heat-induced denaturation/aggregation of soy proteins at low moisture contents. Associated functional properties such as water holding and hydration property can also be evaluated.     

应用差示扫描量热法检测昆虫总蛋白的热滞活性

产生抗冻蛋白是寒带昆虫抵御低温的重要机制之一, 但检测其活性仍存在一些困难, 尤其对于个体较小的昆虫样品。为了探索差示扫描量热法是否适于检测昆虫总蛋白的热滞活性, 本研究利用差示扫描量热法对黄粉虫Tenebrio molitor幼虫的总蛋白和血淋巴分别进行了热滞活性检测。结果表明： 黄粉虫总蛋白的热滞活性（0.49~0.98℃）要低于血淋巴（2.54~4.34℃）。通过这种方法, 进一步检测了3种在内蒙古大兴安岭林区采集到的越冬昆虫： 稠李巢蛾Yponomeuta evonymallus幼虫、 舞毒蛾Lymantria dispar卵和落叶松八齿小蠹Ips subelongatus成虫。结果发现, 它们都存在热滞活性, 其中稠李巢蛾的热滞活性为0.34~0.43℃, 舞毒蛾的热滞活性为0.35~0.42℃, 落叶松八齿小蠹的热滞活性为0.37~0.40℃, 说明这3种昆虫能以产生抗冻蛋白的方式作为越冬策略之一。本研究表明通过差示扫描量热法检测昆虫总蛋白是否存在热滞活性来判断抗冻蛋白的存在是可行的。     

Gene expression and characterization of two 2-oxoacid:ferredoxin oxidoreductases from Aeropyrum pernix K1

A hyperthermophilic and aerobic crenarchaeon, Aeropyrum pernix K1, has two sets of genes possibly encoding 2-oxoacid:ferredoxin oxidoreductases. One is encoded in open reading frames (ORFs) ape2126 and ape2128, and the other in ORFs ape1473 and ape1472. The two sets of genes were expressed. The product enzymes, Ape2126/2128 and Ape1473/1472, showed optimal temperatures of 105 and over 110 degrees C, and optimal pHs of 8.5 and 9.0, respectively, using pyruvate as a substrate. Pyruvate, 2-oxobutyrate, and glyoxylate were the best substrates for both enzymes, and additionally Ape1473/1472 was able to act on 2-oxoglutarate, suggesting the enzyme operates in the TCA cycle.     

Heat killing of bacterial spores analyzed by differential scanning calorimetry.

Thermograms of the exosporium-lacking dormant spores of Bacillus megaterium ATCC 33729, obtained by differential scanning calorimetry, showed three major irreversible endothermic transitions with peaks at 56, 100, and 114 degrees C and a major irreversible exothermic transition with a peak at 119 degrees C. The 114 degrees C transition was identified with coat proteins, and the 56 degrees C transition was identified with heat inactivation. Thermograms of the germinated spores and vegetative cells were much alike, including an endothermic transition attributable to DNA. The ascending part of the main endothermic 100 degrees C transition in the dormant-spore thermograms corresponded to a first-order reaction and was correlated with spore death; i.e., greater than 99.9% of the spores were killed when the transition peak was reached. The maximum death rate of the dormant spores during calorimetry, calculated from separately measured D and z values, occurred at temperatures above the 73 degrees C onset of thermal denaturation and was equivalent to the maximum inactivation rate calculated for the critical target. Most of the spore killing occurred before the release of most of the dipicolinic acid and other intraprotoplast materials. The exothermic 119 degrees C transition was a consequence of the endothermic 100 degrees C transition and probably represented the aggregation of intraprotoplast spore components. Taken together with prior evidence, the results suggest that a crucial protein is the rate-limiting primary target in the heat killing of dormant bacterial spores.