The thermal stability of turnip yellow mosaic virus under hydrostatic pressure

The thermal stability of an isometric plant virus, Turnip Yellow Mosaic Virus (TYMV), has been investigated at low and high hydrostatic pressure, using small angle neutron scattering. Contrast variation allowed us to separately observe the structural changes of the protein capsid and the RNA core. The experiments were performed in 0.05M Tris buffer at pD = 8.0 and in 0.05M bis-Tris buffer at pD = 6.0 containing different H₂O/D₂O mixtures (40% and 70% D₂O). It was found that hydrostatic pressure enhances the stability of TYMV. The thermally induced uncoating of RNA as well as structural transitions of the protein capsid are shifted to higher temperature upon increasing the pressure from 5 × 10⁶ Pa to 2 × 10⁸ Pa.     

The effect of high hydrostatic pressure on eukaryotic protein synthesis

The pressure response of two eukaryotic protein synthesizing systems has been characterized. The rabbit reticulocyte system has been tested, both in vivo and in vitro, using endogenous polysomes and polyuridylic acid (poly U). In addition, the poly U-directed polyphenylalanine synthesizing system obtained from wheat germ was utilized. The effect of pressure on eukaryotic protein synthesis has been found to be basically similar to that observed in prokaryotic systems, although the response of the eukaryotic protein synthesizing system is somewhat more complex signifying a greater influence of overlapping reactions. Magnesium was found to affect eukaryotic systems in much the same way as has been reported for prokaryotic systems, i.e., increasing the Mg²⁺ concentration in a protein synthesizing system increases the barotolerance exhibited by that system. Under conditions of high Mg²⁺ concentration, however, extreme (up to 160%) stimulation of protein synthesis at lower pressure levels was observed in the eukaryotic systems. Such high stimulation is not apparent in prokaryotic systems. The poly U-directed wheat germ system exhibited the most barotolerant polypeptide synthesis ever seen in our laboratory. This extreme barotolerance was only slightly decreased when the system was tested at reduced concentrations of magnesium.     

Molecular crowding effects on structure and stability of DNA

Living cells contain a variety of biomolecules including nucleic acids, proteins, polysaccharides, and metabolites as well as other soluble and insoluble components. These biomolecules occupy a significant fraction (20-40%) of the cellular volume. The total concentration of biomolecules reaches 400gL(-1), leading to a crowded intracellular environment referred to as molecular crowding. Therefore, an understanding of the effects of molecular crowding conditions on biomolecules is important to broad research fields such as biochemical, medical, and pharmaceutical sciences. In this review, we describe molecular conditions in the cytoplasm and nucleus, which are totally different from in vitro conditions, and then show the biochemical and biophysical consequences of molecular crowding. Finally, we discuss the effect of molecular crowding on the structure, stability, and function of nucleic acids and the significance of molecular crowding in biotechnology and nanotechnology.     

Hydration pressure and phase transitions of phospholipids: I. Piezotropic approach

Dehydration reduces the main phase transition pressure of phospholipids. An analysis based on the Gibbs-Duhem equation shows how the shift of the transition pressure is correlated to the hydration pressure.By using Fourier transform infrared (FT-IR) spectroscopy we determined the hydration-dependent phase transition pressure. The application of our new approach gives hydration pressure values which agree with the values obtained with the osmotic stress method.     

Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms

DNA polymerases derived from three thermophilic microorganisms, Pyrococcus strain ES4, Pyrococcus furiosus, and Thermus aquaticus, were stabilized in vitro by hydrostatic pressure at denaturing temperatures of 111°C, 107.5°C, and 100°C (respectively). Inactivation rates, as determined by enzyme activity measurements, were measured at 3, 45, and 89 MPa. Half-lives of P. strain ES4, P. furiosus, and T. aquaticus DNA polymerases increased from 5.0, 6.9, and 5.2 minutes (respectively) at 3 MPa to 12, 36, and 13 minutes (respectively) at 45 MPa. A pressure of 89 MPa further increased the half-lives of P. strain ES4 and T. aquaticus DNA polymerases to 26 and 39 minutes, while the half-life of P. furiosus DNA polymerase did not increase significantly from that at 45 MPa. The decay constant for P. strain ES4 and T. aquaticus polymerases decreased exponentially with increasing pressure, reflecting an observed change in volume for enzyme inactivation of 61 and 73 cm³/mol, respectively. Stabilization by pressure may result from pressure effects on thermal unfolding or pressure retardation of unimolecular inactivation of the unfolded state. Regardless of the mechanism, pressure stabilization of proteins could explain the previously observed extension of the maximum temperature for survival of P. strain ES4 and increase the survival of thermophiles in thermally variable deep-sea environments such as hydrothermal vents. Received: September 12, 1997 / Accepted: February 24, 1998     

High-throughput thermal stability assessment of DNA hairpins based on high resolution melting

On the basis of high-resolution melting, a high-throughput approach to measure melting temperatures (T_ms) of short DNA hairpins was developed. With this method, T_ms of thousands of triloop, tetraloop, and pentaloop hairpins involving various loop sequences and various closing base pairs (cbp) were obtained in hours. The stability of triloop hairpins decreased with the change of cbp (5′–3′) in the order of c-g > g-c > t-a ≥ a-t, showing that the cbp of 5′-Pyr-Pur-3′ (Pyr = pyrimidine, Pur = purine) contributed more stability than 5′-Pur-Pyr-3′. For tetraloop hairpins, GNNA, GNAB, and CNNG (N = A, G, C, or T; B = G, C, or T) were found to be highly stable irrespective of the cbp type. TNNA was also stable in both g-c and a-t families, while CGNA only in the c-g family. Pentaloop hairpins of cTGNAGg, cGNYNAg (Y = T or C) and cCGNNAg were exceptionally stable motifs. In most cases, pyrimidine-rich loops were more favorable to stabilize the whole structure than purine-rich ones. The present approach showed a good performance in assessing the thermal stability of large amounts of DNA hairpins comprehensively. These data are useful to understand the sequence dependence of the stability of DNA secondary structures and promising to improve the structure simulation by consummating basic databases.     

Effect of high hydrostatic pressure on the bacterial mechanosensitive channel MscS

We have investigated the effect of high hydrostatic pressure on MscS, the bacterial mechanosensitive channel of small conductance. Pressure affected channel kinetics but not conductance. At negative pipette voltages (corresponding to membrane depolarization in the inside-out patch configuration used in our experiments) the channel exhibited a reversible reduction in activity with increasing hydrostatic pressure between 0 and 900 atm (90 MPa) at 23°C. The reduced activity was characterized by a significant reduction in the channel opening probability resulting from a shortening of the channel openings with increasing pressure. Thus high hydrostatic pressure generally favoured channel closing. Cooling the patch by approximately 10°C, intended to order the bilayer component of the patch by an amount similar to that caused by 50 MPa at 23°C, had relatively little effect. This implies that pressure does not affect channel kinetics via bilayer order. Accordingly we postulate that lateral compression of the bilayer, under high hydrostatic pressure, is responsible. These observations also have implications for our understanding of the adaptation of mechanosensitive channels in deep-sea bacteria.A Proceeding of the 28th Annual Meeting of the Australian Society for Biophysics.     

Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition

Genome-wide mRNA expression profiles of Saccharomyces cerevisiae growing under hydrostatic pressure were characterized. We selected a hydrostatic pressure of 30 MPa at 25 degrees C because yeast cells were able to grow under these conditions, while cell size and complexity were increased after decompression. Functional characterization of pressure-induced genes suggests that genes involved in protein metabolism and membrane metabolism were induced. The response to 30 MPa was significantly different from that observed under lethal conditions because protein degradation was not activated under 30 MPa pressure. Strongly induced genes those that contribute to membrane metabolism and which are also induced by detergents, oils, and membrane stabilizers.     

Cuticle expansion during feeding in the tick Amblyomma hebraeum (Acari: Ixodidae): The role of hydrostatic pressure

Female Amblyomma hebraeum ticks (Acari: Ixodidae) increase their weight ∼10-fold during a ‘slow phase of engorgement’ (7–9 days), and a further 10-fold during the ‘rapid phase’ (12–24 h). During the rapid phase, the cuticle thins by half, with a plastic (permanent) deformation of greater than 40% in two orthogonal directions. A stress of 2.5 MPa or higher is required to achieve this degree of deformation (Flynn and Kaufman, 2015). Using a dimensional analysis of the tick body and applying the Laplace equation, we calculated that the tick must achieve high internal hydrostatic pressures in order to engorge fully: greater than 55 kPa at a fed:unfed mass ratio of ∼20:1, when cuticle thinning commences (Flynn and Kaufman, 2011). In this study we used a telemetric pressure transducer system to measure the internal hydrostatic pressure of ticks during feeding. Sustained periods of irregular high frequency (>20 Hz) pulsatile bursts of high pressure (>55 kPa) were observed in two ticks: they had been cannulated just prior to the rapid phase of engorgement, and given access to a host rabbit for completion of the feeding cycle. The pattern of periods of high pressure generation varied over the feeding cycle and between the two specimens. We believe that these pressures exceed those reported so far for any other animal.     

On the effect of hydrostatic pressure on the conformational stability of globular proteins

The model developed for cold denaturation (Graziano, PCCP 2010, 12, 14245‐14252) is extended to rationalize the dependence of protein conformational stability upon hydrostatic pressure, at room temperature. A pressure− volume work is associated with the process of cavity creation for the need to enlarge the liquid volume against hydrostatic pressure. This contribution destabilizes the native state that has a molecular volume slightly larger than the denatured state due to voids existing in the protein core. Therefore, there is a hydrostatic pressure value at which the pressure−volume contribution plus the conformational entropy loss of the polypeptide chain are able to overwhelm the stabilizing gain in translational entropy of water molecules, due to the decrease in water accessible surface area upon folding, causing denaturation. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 711–718, 2015.     

The effects of polyols on the thermal stability of calf thymus DNA

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, (−) and (+) arabitol, -mannitol, -sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (Δ_dH) and temperature of the maximum of the denaturation peak (T_max) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of (−) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for (−) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of (−) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.     

The stability of rabbit reticulocyte polysomes at high hydrostatic pressure

The application of high hydrostatic pressure to an in vitro rabbit reticulocyte, polypeptide-synthesizing system has been shown to inhibit synthesis either partially or totally depending upon the magnitude of pressure utilized (Scheck, A.C. and Landau, J.V. (1982) Biochim. Biophys. Acta 718, 21–25). This paper shows that the total inhibition of synthesis seen at 670 atm is similar to the inhibition of elongation produced by cycloheximide in that the polysome profiles remain intact. Partial inhibition at 300 atm shows a reduced rate of ribosome run-off with elongation being affected to the same, or greater extent than initiation. In no instance was disassociation of polysomes seen as a causative factor in the inhibition of synthesis by high pressure.     

Influence of formamide on the thermal stability of DNA

The utility of formamide in the denaturation and renaturation of DNA has been examined. The melting temperature of duplex DNA is lowered by 0·6°C per per cent formamide. The depression of melting temperature is independent of the GC content. Formamide also increases the width of the thermal transition. Upto 30%, it does not affect the rate of DNA reassociation     

The effect of pressure on the glass transition of biopolymer/co-solute. Part I: The example of gelatin

High-solid materials of gelatin in the presence of co-solute were prepared and subjected to a series of hydrostatic pressures up to 700 MPa. Following this, a study was made of the relaxation properties of the mixture around the glass transition region and the melting behaviour of the gelatin network. Structural properties were monitored using differential scanning calorimetry and small-deformation dynamic oscillation on shear. Thermograms were obtained and master curves of viscoelasticity were constructed for each experimental pressure. The dependence of the empirical shift distances obtained from mechanical measurements and supplementing evidence from thermal analysis argue that the application of pressure did not alter the vitrification or melting characteristics of the gelatin/co-solute system within the experimentally accessible pressure range. Unlike the principle of the time–temperature–pressure superposition applicable to synthetic macromolecules, it may not be possible to incorporate a pressure component into the framework of thermorheological simplicity governing the glass transition of the high-sugar gelatin network.     

Cationic effect of imidazolium-based ionic liquid on the stability of myoglobin

The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM ILs with the same cation 1-butyl-3-methylimidazolium (Bmim⁺) but different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of ILs in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim⁺ cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of ILs increases with increasing alkyl chain length of imidazolium cation of ILs and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.     

Role of nitric oxide in the response of Saccharomyces cerevisiae cells to heat shock and high hydrostatic pressure

Nitric oxide (NO) is a simple and unique molecule that has diverse functions in organisms, including intracellular and intercellular messenger. The influence of NO on cell growth of Saccharomyces cerevisiae and as a signal molecule in stress response was evaluated. Respiring cells were more sensitive to an increase in intracellular NO concentration than fermentatively growing cells. Low levels of NO demonstrated a cytoprotective effect during stress from heat-shock or high hydrostatic pressure. Induction of NO synthase was isoform-specific and dependent on the metabolic state of the cells and the stress response pathway. These results support the hypothesis that an increase in intracellular NO concentration leads to stress protection.     

Effect of diethylsulfoxide on the thermal denaturation of DNA

DNA thermal denaturation has been investigated in aqueous solutions of diethylsulfoxide (DESO) by means of UV-vis and densimetry methods. It is suggested that, on the one hand, the structural change of entire solutions and, on the other hand, a direct interaction of DESO with DNA are responsible for the observed peculiar behavior. The results obtained were compared with those of dimethylsulfoxide (DMSO), also known from literature.     

The effects of hydrostatic pressure on the low-K m GTPase in brain membranes from two congeneric marine fishes

To investigate, the effects of hydrostatic pressure on transmembrane signaling in cold-adapted marine fishes, we examined the high-affinity GTPase activity in two congeneric marine fishes, Sebastolobus alascanus and S. altivelis. In brain membranes there are two GTPase activities, one with a low K _m and one with a high K _m for GTP. The high-affinity GTPase activity, characteristic of the subunits of the guanine nucleotide binding protein pool, was stimulated by the A₁ adenosine receptor agonists N ⁶(R-phenylisopropyl)adenosine and N ⁶-cyclopentyladenosine, and the muscarinic cholinergic agonist carbamyl choline. Pertussis toxin-catalyzed ADP-ribosylation of the membranes for 2 h at 5°C prior to the GTPase assay decreased the basal GTPase activity 30–40% and abolished N ⁶ (R-phenylisopropyl)adenosine stimulation of GTP hydrolysis. Basal high-affinity hydrolysis of GTP, measured at 0.3 mol·1^-1GTP, was stimulated 22% in both species by 340 atm pressure. At 340 atm pressure, the apparent K _m of GTP is decreased approximately 10% in each of the species, and the V _max values are increased 11 and 15.9% in S. alascanus and S. altivelis, respectively. The apparent volume changes associated with the decreased K _m of GTP and the increased V _max ranged from-7.0 to-9.9 ml·mol^-1. Increased pressure markedly decreased the efficacy of N ⁶ (R-phenylisopropyl) adenosine, N ⁶-cylcopentyladenosine and carbamyl choline in stimulating GTPase activity. The effects of increased hydrostatic pressure on transmembrane signal transduction by the A₁ adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system may stem, at least in part, from pressure-increased GTP hydrolysis and the concomitant termination of inhibitory signal transduction.Abbreviations [³H] DPCPX ³H cyclopentyl-1, 3-dipropylxanthine - AppNHp 5-adenylylimidodiphosphate - cpm counts per minute - CPA N ⁶-cyclopentyladenosine - EDTA ethylenediaminetetra acetic acid - EGTA ethyleneglycol-bis (-aminoethylether) N, N, N, N-totra-acctic acid - G protein guanine nucleotide binding protein - G_i inhibitory G protein - G_o other G protein, common in brain membranes - G_s stimulatory G protein - GTPase guanosine triphosphatase - K _i inhibition constant - K _m Michaelis constant - pK _a log of the dissociation constant - R-PIA N ⁶ (R-phenylisopropyl) adenosine - TRIS tris[hydroxymethyl]aminomethane - V_max maximal velocity - [-³²P]GTP [-³²P] guanosine 5-triphosphate (tetra (triethylammonium) salt)     

Singular value decomposition of 3-D DNA melting curves reveals complexity in the melting process

The thermal denaturation of synthetic deoxypolynucleotides of defined sequence was studied by a three dimensional melting technique in which complete UV absorbance spectra were recorded as a function of temperature. The results of such an experiment defined a surface bounded by absorbance, wavelength, and temperature. A matrix of the experimental data was built, and analyzed by the method of singular value decomposition (SVD). SVD provides a rigorous, model-free analytical tool for evaluating the number of significant spectral species required to account for the changes in UV absorbance accompany-ing the duplex – to – single strand transition. For all of the polynucleotides studied (Poly dA – Poly dT; [Poly (dAdT)]₂; Poly dG – Poly dC; [Poly(dGdC)]₂), SVD indicated the existence of at least 4 – 5 significant spectral species. The DNA melting transition for even these simple repeating sequences cannot, therefore, be a simple two-state process. The basis spectra obtained by SVD analysis were found to be unique for each polynucleotide studied. Differential scanning calorimetry was used to obtain model free estimates for the enthalpy of melting for the polynucleotides studied, with results in good agreement with previously published values. Received: 16 April 1997 / Accepted: 9 July 1997     

Synergistic effects of atmospheric pressure plasma‐emitted components on DNA oligomers: a Raman spectroscopic study

Cold atmospheric‐pressure plasmas have become of increasing importance in sterilization processes especially with the growing prevalence of multi‐resistant bacteria. Albeit the potential for technological application is obvious, much less is known about the molecular mechanisms underlying bacterial inactivation. X‐jet technology separates plasma‐generated reactive particles and photons, thus allowing the investigation of their individual and joint effects on DNA. Raman spectroscopy shows that particles and photons cause different modifications in DNA single and double strands. The treatment with the combination of particles and photons does not only result in cumulative, but in synergistic effects. Profilometry confirms that etching is a minor contributor to the observed DNA damage in vitro.

Schematics of DNA oligomer treatment with cold atmospheric‐pressure plasma.