Thermal Renaturation Research Articles

NanoLuc tandem repeats show misfolding during thermal denaturation which is reversible by E. coli chaperones (DnaK/DnaJ/GrpE).

Monomeric NanoLuc (Nluc) is a thermally robust bioluminescent protein with its thermal denaturation temperature at 58oC. In order to further examine this thermal behavior, we developed three protein constructs (monomeric, dyad, and triad Nluc). Interestingly, thermal denaturation experiments at 58oC showed sudden decrease in thermal stability when Nluc was linked to itself, dyad and triad Nluc constructs, while monomeric Nluc remained mostly folded under same conditions. Also, all three constructs maintained their activity (bioluminescence) when brought back to room temperature with no spontaneous recovery observed. However, when we examined the E. coli DnaK/DnaJ/GrpE chaperone system effect on this loss of thermal stability in refolding assays, we observed recovery in bioluminescence signal up to 70% for the poly-Nluc constructs. This observation strongly supports poly-Nluc is a strong chaperone substrate. Another interesting observation was that the refolding of these poly-Nluc constructs was possible at similar percentages even in the absence of GrpE showing a deviation from the canonical chaperone mechanism described up to now in literature. However, high GrpE concentrations were inhibitory to the protein refolding. Similarly, addition of disaggregase ClpB chaperone, showed no further assistance in refolding of the proteins and in some cases proved to be inhibiting the refolding. Lastly, we performed MD simulations of thermally unfolded and refolded monomeric and poly-Nluc constructs. These show that monomeric Nluc is able to correctly refold, while dyad Nluc misfolds with 78% and triad Nluc misfolds with 63%. Also, additional MD simulations suggest the reason of dyad and triad Nluc constructs misfolding is due to domain swapping preventing the correct refolding of these two constructs during thermal renaturation.

Thermal renaturation of rivers in the post-industrial age - An example of the Przemsza River basin (Poland)

This article concerns temperature, which is one of the most important physical properties of surface waters. During the period of climate warming, tendencies of increasing river temperature have been repeatedly identified in the literature. This article discusses the lowering of the river temperature, a phenomenon rarely considered, which is occurring in a heavily industrialised area in the southern part of Poland (Upper Silesia Region), undergoing deep restructuring.The main aim of the study is to analyse and evaluate the unique Przemsza River basin, which differs from other river basins in terms of the thermal regime of its rivers and its tendencies for change. These changes are presented through the long period of 1961–2015, and additionally in two sub-periods, 1961–1994 and 1995–2015, differing with respect to the degree of the organisation of wastewater management. This was possible to demonstrate thanks to the high density of hydrological stations within a small area, which is rare, especially over such a long period. This work, therefore, fills the research gap on changes in the thermal regime of rivers caused by water management. Its results differ from those presented so far in the literature and expand the knowledge of this subject.In the years under consideration (1961–2015), the air temperature increased by 0.03 °C·per year−1 on average. It was found that the temperature of rivers (or their sections) decreased by as much as −0.8 °C·year−1. The greatest drops in temperature occurred in the winter season and amounted to a maximum of −0.12 °C·season−1. In the first part of the analysed period (1961–1994), unnaturally high river temperatures caused by water management factors were recorded. In turn, in the years 1995–2015 there was a decrease in temperature caused by various technical operations carried out in the catchments, including the construction of modern sewage treatment plants, as well as limitation of the introduction of water into rivers from closed hard coal mines. This phenomenon is referred to as thermal renaturation. Maintaining positive changes in the thermal regime requires, inter alia, a further reduction in the amount of pollutants discharged into rivers by water discharges from mine drainage.

Detection of Thermal Denaturation and Renaturation of Collagen: Optical Characteristics Using Angular Displacement-Enhanced Heterodyne Polarimeter

An angular displacement-enhanced heterodyne polarimeter has been employed to investigate the optical properties and thermal denaturation of collagen. In the angular measurement, the phase change of the collagen at different temperatures can be enhanced using a null-detection technique with a retardation-tunable wave-plate and analyzer into our common-path heterodyne polarimeter. Also, the environmental noise can be reduced by the interference of transverse magnetic (TM) and transverse electric (TE) waves from the optical common-path setup. We first measured the optical rotation of deionized water, glucose solution (dextrorotatory), and bovine serum albumin (BSA, levorotatory) to ensure the accuracy and high signal-to-noise ratio (SNR) of the experimental setup. It was found that the optical rotation from the measured phase difference can be enhanced up to 40-fold amplification with a soleil babinet compensator (SBC). Moreover, we have investigated the thermal denaturation from the denaturation temperature defined by the change of optical rotation, and the thermal renaturation of collagen has been studied by heating the samples to 55°C followed by cooling down to 25°C and then heating back to 55°C. The results reveal that 63% triple helices in the collagen structure have been refolded during the renaturation stage. This approach successfully provides with a real-time, noninvasive, and high resolution detection for thermal denaturation and renaturation of collagen.

Rigid DNA Beams for High‐Resolution Single‐Molecule Mechanics

Rigid DNA Beams for High‐Resolution Single‐Molecule Mechanics

Nocardioides ultimimeridianus sp. nov. and Nocardioides maradonensis sp. nov., isolated from rhizosphere soil

Two novel Gram-reaction-positive, rod-shaped actinobacterial strains, designated RP-B26(T) and RP-B30(T), were isolated from rhizosphere soil of a cliff-associated plant (Peucedanum japonicum Thunb.) which was collected from Mara Island, Republic of Korea. The colonies of the isolates were circular, smooth, convex and moderately yellow-light-yellow in colour. 16S rRNA gene sequence analysis revealed that the isolates belonged to the family Nocardioidaceae and formed two distinct sublineages within the radiation of the genus Nocardioides. 16S rRNA gene sequence similarity between the isolates was 98.2 %. The closest phylogenetic neighbours of strain RP-B26(T) were Nocardioides humi DCY24(T) and Nocardioides kongjuensis A2-4(T) with 97.4 and 97.0 % 16S rRNA gene sequence similarity, respectively, whereas 16S rRNA gene sequence similarities between strain RP-B30(T) and N. humi DCY24(T) and N. kongjuensis A2-4(T) were 96.5 and 96.0 %, respectively. Both of the isolates contained ll-diaminopimelic acid as the diagnostic diamino acid in the cell walls. The predominant menaquinone was MK-8(H(4)). The polar lipids were phosphatidylinositol and phosphatidylglycerol. The fatty acid profiles of the isolates were characterized by the presence of saturated, unsaturated, 10-methyl and hydroxyl fatty acids, with small amounts of branched fatty acids. The DNA G+C contents of strains RP-B26(T) and RP-B30(T) were 73.0 and 71.7 mol%, respectively. Levels of DNA-DNA relatedness between the isolates were 44.9 ± 1.5 % (thermal renaturation method) and 43.2 % (photobiotin-labelled method); the isolates showed low DNA-DNA relatedness values (<11 %) to the most closely related strain, N. humi KCTC 19265(T). On the basis of the phenotypic, genotypic and DNA-DNA hybridization data presented here, the isolates are considered to represent two novel species of the genus Nocardioides, for which the names Nocardioides ultimimeridianus sp. nov. (type strain RP-B26(T) = KCTC 19368(T) = DSM 19768(T)) and Nocardioides maradonensis sp. nov. (type strain RP-B30(T) = KCTC 19384(T) = DSM 19769(T)) are proposed.

Mechanism of Thermal Renaturation and Hybridization of Nucleic Acids: Kramers’ Process and Universality in Watson−Crick Base Pairing

Renaturation and hybridization reactions lead to the pairing of complementary single-stranded nucleic acids. We present here a theoretical investigation of the mechanism of these reactions in vitro under thermal conditions (dilute solutions of single-stranded chains, in the presence of molar concentrations of monovalent salts and at elevated temperatures). The mechanism follows a Kramers' process, whereby the complementary chains overcome a potential barrier through Brownian motion. The barrier originates from a single rate-limiting nucleation event in which the first complementary base pairs are formed. The reaction then proceeds through a fast growth of the double helix. For the DNA of bacteriophages T7, T4, and phiX174, as well as for Escherichia coli DNA, the bimolecular rate k2 of the reaction increases as a power law of the average degree of polymerization <N> of the reacting single-strands: k2 is proportional to <N> alpha. This relationship holds for 100 < or = <N> < or = 50,000 with an experimentally determined exponent alpha = 0.51 +/- 0.01. The length dependence results from a thermodynamic excluded-volume effect. The reacting single-stranded chains are predicted to be in universal good solvent conditions, and the scaling law is determined by the relevant equilibrium monomer contact probability. The value theoretically predicted for the exponent is alpha = 1 - nutheta2, where nu is Flory's swelling exponent (nu approximately equal 0.588), and theta2 is a critical exponent introduced by des Cloizeaux (theta2 approximately equal 0.82), yielding alpha = 0.52 +/- 0.01, in agreement with the experimental results.

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA(Met) (hmtRNA(Met)) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA(Met) with a C8. The hmtRNA(Met) serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U-->C mutation leads to a failure of the tRNA to respond conformationally to Mg(2+). This mutation results in a drastic disruption of the structure of the hmtRNA(Met), which significantly reduces its aminoacylation. The small fraction of hmtRNA(Met) that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg(2+), the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg(2+), the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg(2+)-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.

Scanning Force Microscopy Study on a Single-Stranded DNA: The Genome of Parvovirus B19

The genome of parvovirus B19 is a 5600-base-long single-stranded DNA molecule with peculiar sequence symmetries. Both complementary forms of this single-stranded DNA are contained in distinct virions and they hybridize intermolecularly to double-stranded DNA if extracted from the capsids with traditional methods, thus losing some of their native structural features. A scanning force microscopy analysis of these double-stranded DNA molecules after thermal denaturation and renaturation gave us the chance to study the possible states that this DNA can assume in both its single-stranded and double-stranded forms. A novel but still poorly reproducible in situ lysis experiment that we have conducted on single virions with the scanning force microscope made it possible to image the totally unpaired state that the single-stranded DNA molecule most likely assumes inside the viral particle. Structural considerations on single molecules offer the opportunity for the formulation of plausible hypotheses on the interaction between the DNA and the viral structural proteins that could prove important for the DNA packaging in the capsid and, possibly, the viral infection mechanisms.

Strong reproductive isolation between closely related tropical sea urchins (genus Echinometra).

Morphological, mitochondrial DNA, and single-copy nuclear DNA differences show that the tropical sea urchin Echinometra mathaei is composed of at least four independent gene pools. Evolutionary distance between species measured with restriction-site changes (for mitochondrial DNA) and thermal renaturation (for single-copy nuclear DNA) is 1%-3% nucleotide divergence. Thus these are the most closely related sea urchin species known. Despite this genetic similarity, strong blocks to interspecific fertilization exist in this genus. Between two Hawaiian species, few eggs are fertilized in hybrid crosses, even in the presence of excess sperm. Microscopic examination of such crosses shows that sperm attachment to heterologous eggs is inhibited. Measures of genetic distance between species can help reveal the tempo of speciation and allow comparisons of morphological, biochemical, and ecological characteristics to be made in an evolutionary framework. Our results show that strong reproductive isolation can evolve by changes in egg-sperm recognition without extensive genetic divergence between species. Such mechanisms are most easily studied in free-spawning animals such as sea urchins but as well may represent an important aspect of speciation in species with internal fertilization.

Rates of molecular evolution and the fraction of nucleotide positions free to vary

Selective constraints on DNA sequence change were incorporated into a model of DNA divergence by restricting substitutions to a subset of nucleotide positions. A simple model showed that both mutation rate and the fraction of nucleotide positions free to vary are strong determinants of DNA divergence over time. When divergence between two species approaches the fraction of positions free to vary, standard methods that correct for multiple mutations yield severe underestimates of the number of substitutions per site. A modified method appropriate for use with DNA sequence, restriction site, or thermal renaturation data is derived taking this fraction into account. The model also showed that the ratio of divergence in two gene classes (e.g., nuclear and mitochondrial) may vary widely over time even if the ratio of mutation rates remains constant. DNA sequence divergence data are used increasingly to detect differences in rates of molecular evolution. Often, variation in divergence rate is assumed to represent variation in mutation rate. The present model suggests that differing divergence rates among comparisons (either among gene classes or taxa) should be interpreted cautiously. Differences in the fraction of nucleotide positions free to vary can serve as an important alternative hypothesis to explain differences in DNA divergence rates.

Purification and characterization of glia maturation factor beta: a growth regulator for neurons and glia.

A protein has been isolated from bovine brains by using a modification of the procedure used to purify glia maturation factor. The method consists of ammonium sulfate precipitation, chromatography with DEAE-Sephacel, Sephadex G-75, and hydroxylapatite columns, passage through a heparin-Sepharose column, and finally fractionation by reverse-phase HPLC with a C4 column. The isolated protein reacts strongly with the mouse monoclonal antibody G2-09 and has a molecular weight of approximately 17,000 and an isoelectric point of pH 4.9. The N terminus is blocked, but tryptic digestion releases 28 peptides, 8 of which have been sequenced. The total known residues add up to more than two-thirds of the entire 140-residue protein, estimated from amino acid composition, and show no sequence homology with any known protein. Reversible thermal renaturation greatly enhances its biological activity. The purified protein stimulates differentiation of normal neurons as well as glial cells. It inhibits the proliferation of the N-18 neuroblastoma line and the C6 glioma line while promoting their phenotypic expression. We designate this protein glia maturation factor beta.

The effect of nitroheterocyclic drugs on DNA: An in vitro model of cytotoxicity

Electrolytic reduction of several nitroheterocyclic drugs was carried out at controlled potentials under anoxic (anaerobic) conditions in the presence of Esherichia coli DNA. The DNA was examined during the reduction process by viscometry, determination of T m values, and thermal renaturation of DNA; its single strand content was determined using hydroxyapatite chromatography and its electrophoretic migration characteristics in acridine-impregnated agarose gels. All the drugs examined decreased the viscosity, thermal renaturation and T m value, and increased the single strand content and migration distance of DNA. The results indicate that the drugs cause strand breakage of DNA as a primary mechanism of action, resulting in extensive loss of helix formation and a concomitant decrease in molecular weight. The method of drug reduction and the techniques to assess DNA damage may be used as a model to determine the relative cytotoxicities and the potential of such agents as antimicrobial and radiosensitizing drugs.

Intrastrand self-complementary sequences in Bacillus subtilis DNA.

Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin kieselguhr (MAK). The instrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulse-labelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.

Variation in satellite DNA from some higher plants.

Pure satellite DNAs were prepared as minor components after centrifugation to equilibrium on CsCl gradients. A single satellite DNA band was isolated from flax (Linum usitatissimum) DNA and two bands were resolved in cucumber (Cucumis sativus) DNA. These apparently homogeneous components of the plant genomes were further analyzed by thermal denaturation and renaturation. The flax satellite DNA appeared homogeneous on thermal denaturation but was shown to contain several components of renaturation. The two cucumber satellite DNAs were different from each other, but both showed at least two components in denaturation and renaturation analyses. Renaturation in the three satellites, particularly in flax, was inaccurate, indicating a considerable degree of sequence divergence. Although each satellite contained quite large amounts of simple repetitious sequences, a residual heterogeneous DNA fraction was always present. It is considered that this was too large a portion of the satellite DNA to be due to organelle or ribosomal DNA in cucumber. The latter possibility is precluded in flax, where the satellite is completely resolved in buoyant density from both organelle and ribosomal DNA.

Polyacrylamide gel as a medium for DNA dissociation and reassociation.

Several properties of thermal denaturation and renaturation of DNA in polyacrylamide gels were investigated: (1) Following electrophoresis the DNA band was scanned and shown to increase in absorbance with increasing temperature. The increase was proportioned to DNA concentration across the peak. (2) The dependence of the Tm on salt concentration over a hundred fold range was similar to that found for DNA in free solution. (3) Denaturation of several DNA samples ranging in G + C content from 26 to 71% was compared in gels and free solution. The relationship between Tm and % G + C was virtually identical for both sets of DNAs. (4) The kinetics of DNA renaturation in the gel was followed. Reassociation of bacteriophage T4 DNA was 2nd order and proceeded more rapidly in polyacrylamide gels than in free solution.

The chloroplastic DNA of Chlorella pyrenoidosa (Emerson strain): heterogeneity and complexity.

The heterogeneity and the complexity of Emerson strain Chlorella pyrenoidosa chloroplastic DNA have been investigated by means of thermal denaturation and renaturation kinetics, and the results have been compared with those of the strain 211/8b of the same alga. The thermal denaturation properties are very close to those of the other strain: the Tm of 65 degrees C in 0.1 standard saline citrate, the maximal hyperchromicity of 41%, and the dispersion coefficent delta 2/3 of 6.65 degrees C. The first derivated curves of the melting profiles show also five components. Denatured chloroplastic DNA renatures rapidly. Two fractions are found; their kinetic complexities have been estimated: 1.5 times 10(7) daltons for the fast renaturing fraction; 2x 10(8) daltons for the low d (G + C) content of the chloroplastic DNA: 1.24 times 10(8) daltons). The unique nucleotide sequence is present in about 19 copies per chloroplastic genome. This report confirms the homogeneity of the chloroplastic genome of algae.

Chromatography of nucleic acids on polynucleotide-coated kieselguhr

This paper describes a new type of fractionating column for nucleic acids. The method is based upon dioxane-dependent binding of nucleic acids to particles coated with the hexammine cobalt salt of a synthetic or natural polynucleotide. This interaction occurs rapidly in the cold, at low ionic strength and apparently without stringent requirements as to sequence complementarity between the interacting pairs. Preliminary observations indicating the general applicability of this method to nucleic acids of microbial and animal origin led to detailed observations on the chromatography of denatured DNA's of bacterial origin. The separation of complementary strands of the denatured DNA of Escherichia coli was obtained on columns containing poly I. The two separated fractions were characterized by temperature-absorbance studies. The fractions were subjected to thermal renaturation alone, mixed together, and mixed with each of two complementary groups of the same DNA specimen fractionated on columns of methylated albumin kieselguhr. The first fraction contained more guanine than cytosine, and the second fraction showed a complementary base composition. Neither of these fractions could re-anneal alone, but renaturation took place when they were mixed together. Each fraction could form hybrids with only one of the products of the methylated albumin kieselguhr column; the two fractions re-annealed with different products of the second column. The resolution of denatured DNA of Micrococcus lysodeikticus and Xanthomonas oryzae, respectively, into two components by means of chromatography on columns containing poly I was also obtained. The chromatography of RNA of microbial origin on columns bearing an animal DNA was studied, employing gel penetration, sedimentation and radioactive assay methods to characterize the products. Partial separations of unlabelled tRNA and 14C-labelled rRNA, and of total tRNA and [ 14C]Val-tRNA, were seen; the concentration of the radioactive RNA's was at best 2-fold. A more effective fractionation was obtained with a mixture of a low-molecular-weight RNA and a rapidly sedimenting rRNA.

Studies of the binding of ethidium bromide to transfer ribonucleic acid: Absorption, fluorescence, ultracentrifugation and kinetic investigations

Ethidium bromide forms a complex with Escherichia coli transfer ribonucleic acid that exhibits many similarities to the deoxyribonucleic acid-ethidium bromide complex. Evidence for the strong interaction is obtained from ultracentrifugation, absorption and fluorescence studies and thermal denaturation profiles. The existence of strong and weak binding sites is indicated by spectrophotometry and spectrofluorometry. The reversibility of complex formation is shown by thermal renaturation studies and by the kinetics of the dissociation, from which it is concluded that several forms of the complex are present at equilibrium. The kinetics of the association reaction reveal the presence of a fast binding process followed by several slow unimolecular steps.

The nature of strong binding between acridine orange and deoxyribonucleic acid as revealed by equilibrium dialysis and thermal renaturation

The strong binding of acridine orange with DNA was studied by means of equilibrium dialysis and thermal renaturation of the complex. Thermodynamic parameters for the strong binding with native and denatured DNA were calculated from the data of equilibrium dialysis. The association constant and the change of free energy were nearly equal for both types of DNA, but the changes of enthalpy and entropy were quite different. Binding to both types of DNA may be strong binding in nature, but the accompanying effects on the secondary structures of DNA would be different. It was found that the presence of acridine orange inhibits the completion of renaturation, presumably due to a stabilizing effect of the bound dye on the “wrong” base pairs as they are formed. The results support the notion that binding of acridine orange with the double helix is an intercalation into adjacent base pairs, while the binding with single strand is a partial intercalation into adjacent bases.

Interaction of lysine-rich histones and DNA

Complexes of lysine-rich histones (f1) with native linear calf thymus DNA were prepared in soluble form by a salt-gradient dialysis method and examined by a variety of physico-chemical methods. Absorption spectroscopy and circular dichroism revealed minimal differences between DNA and histone-DNA complexes, suggesting that the DNA remains in the B conformation with histone present. Thermal denaturation and thermal renaturation studies indicated that the presence of histone resulted in a marked stabilization of DNA structure and permitted rapid renaturation of denatured regions. Maximum renaturation was effected with as few as 20 to 40 histone molecules per DNA molecule (10 7 mol. wt). Sedimentation velocity measurements of the complexes indicated that, at infinite dilution, the particles behave as single DNA molecules with associated histones. Evidence for concentration-dependent aggregation of the histone-DNA particles was also obtained. Dye-binding studies suggested that lysine-rich histone does not bind in the small groove of DNA; the presence of histone on DNA does not inhibit actinomycin binding. Furthermore, evidence was obtained indicating that histone might bind within the large groove of DNA; histone complexes with unglucosylated T2 ∗ DNA were less effective substrates for glucosylation by α-glucosyl transferase than was T2 ∗ DNA.