Pressure-induced Denaturation Research Articles

Trimethylamine-N-oxide depletes urea in a peptide solvation shell

Trimethylamine-N-oxide (TMAO) and urea are metabolites that are used by some marine animals to maintain their cell volume in a saline environment. Urea is a well-known denaturant, and TMAO is a protective osmolyte that counteracts urea-induced protein denaturation. TMAO also has a general protein-protective effect, for example, it counters pressure-induced protein denaturation in deep-sea fish. These opposing effects on protein stability have been linked to the spatial relationship of TMAO, urea, and protein molecules. It is generally accepted that urea-induced denaturation proceeds through the accumulation of urea at the protein surface and their subsequent interaction. In contrast, it has been suggested that TMAO's protein-stabilizing effects stem from its exclusion from the protein surface, and its ability to deplete urea from protein surfaces; however, these spatial relationships are uncertain. We used neutron diffraction, coupled with structural refinement modeling, to study the spatial associations of TMAO and urea with the tripeptide derivative glycine-proline-glycinamide in aqueous urea, aqueous TMAO, and aqueous urea-TMAO (in the mole ratio 1:2 TMAO:urea). We found that TMAO depleted urea from the peptide's surface and that while TMAO was not excluded from the tripeptide's surface, strong atomic interactions between the peptide and TMAO were limited to hydrogen bond donating peptide groups. We found that the repartition of urea, by TMAO, was associated with preferential TMAO-urea bonding and enhanced urea-water hydrogen bonding, thereby anchoring urea in the bulk solution and depleting urea from the peptide surface.

An X-ray and Neutron Scattering Study of Aqueous MgCl2 Solution in the Gigapascal Pressure Range

The structure of electrolyte solutions under pressure at a molecular level is a crucial issue in the fundamental science of understanding the nature of ion solvation and association and application fields, such as geological processes on the Earth, pressure-induced protein denaturation, and supercritical water technology. We report the structure of an aqueous 2 m (=mol kg−1) MgCl2 solution at pressures from 0.1 MPa to 4 GPa and temperatures from 300 to 500 K revealed by X-ray- and neutron-scattering measurements. The scattering data are analyzed by empirical potential structure refinement (EPSR) modeling to derive the pair distribution functions, coordination number distributions, angle distributions, and spatial density functions (3D structure) as a function of pressure and temperature. Mg2+ forms rigid solvation shells extended to the third shell; the first solvation shell of six-fold octahedral coordination with about six water molecules at 0 GPa transforms into about five water molecules and one Cl− due to the formation of the contact ion pairs in the GPa pressure range. The Cl− solvation shows a substantial pressure dependence; the coordination number of a water oxygen atom around Cl− increases from 8 at 0.1 MPa/300 K to 10 at 4 GPa/500 K. The solvent water transforms the tetrahedral network structure at 0.1 MPa/300 K to a densely packed structure in the GPa pressure range; the number of water oxygen atoms around a central water molecule gradually increases from 4.6 at 0.1 MPa/298 K to 8.4 at 4 GPa/500 K.

Hysteretic Pressure Dependence of Ca2+ Binding in LH1 Bacterial Membrane Chromoproteins

Much of the thermodynamic parameter values that support life are set by the properties of proteins. While the denaturing effects of pressure and temperature on proteins are well documented, their precise structural nature is rarely revealed. This work investigates the destabilization of multiple Ca2+ binding sites in the cyclic LH1 light-harvesting membrane chromoprotein complexes from two Ca-containing sulfur purple bacteria by hydrostatic high-pressure perturbation spectroscopy. The native (Ca-saturated) and denatured (Ca-depleted) phases of these complexes are well distinguishable by much-shifted bacteriochlorophyll a exciton absorption bands serving as innate optical probes in this study. The pressure-induced denaturation of the complexes related to the failure of the protein Ca-binding pockets and the concomitant breakage of hydrogen bonds between the pigment chromophores and protein environment were found cooperative, involving all or most of the Ca2+ binding sites, but irreversible. The strong hysteresis observed in the spectral and kinetic characteristics of phase transitions along the compression and decompression pathways implies asymmetry in the relevant free energy landscapes and activation free energy distributions. A phase transition pressure equal to about 1.9 kbar was evaluated for the complexes from Thiorhodovibrio strain 970 from the pressure dependence of biphasic kinetics observed in the minutes to 100 h time range.

Ligand-Dependent Volumetric Characterization of Manganese Riboswitch Folding: A High-Pressure Single-Molecule Kinetic Study.

Nanoscopic differences in free volume result in pressure-dependent changes in free energies which can therefore impact folding/unfolding stability of biomolecules. Although such effects are typically insignificant under ambient pressure conditions, they are crucially important for deep ocean marine life, where the hydraulic pressure can be on the kilobar scale. In this work, single molecule FRET spectroscopy is used to study the effects of pressure on both the kinetics and overall thermodynamics for folding/unfolding of the manganese riboswitch. Detailed pressure-dependent analysis of the conformational kinetics allows one to extract precision changes (σ ≲ 4-8 Å3) in free volumes not only between the fully folded/unfolded conformations but also with respect to the folding transition state of the manganese riboswitch. This permits first extraction of a novel "reversible work" free energy (PΔV) landscape, which reveals a monotonic increase in manganese riboswitch volume along the folding coordinate. Furthermore, such a tool permits exploration of pressure-dependent effects on both Mn2+ binding and riboswitch folding, which demonstrate that ligand attachment stabilizes the riboswitch under pressure by decreasing the volume increase upon folding (ΔΔV < 0). Such competition between ligand binding and pressure-induced denaturation dynamics could be of significant evolutionary advantage, compensating for a weakening in riboswitch tertiary structure with pressure-mediated ligand binding and promotion of folding response.

Hydrostatic High-Pressure-Induced Denaturation of LH2 Membrane Proteins.

The denaturation of globular proteins by high pressure is frequently associated with the release of internal voids and/or the exposure of the hydrophobic protein interior to a polar aqueous solvent. Similar evidence with respect to membrane proteins is not available. Here, we investigate the impact of hydrostatic pressures reaching 12 kbar on light-harvesting 2 integral membrane complexes of purple photosynthetic bacteria using two types of innate chromophores in separate strategic locations: bacteriochlorophyll-a in the hydrophobic interior and tryptophan at both protein-solvent interfacial gateways to internal voids. The complexes from mutant Rhodobacter sphaeroides with low resilience against pressure were considered in parallel with the naturally robust complexes of Thermochromatium tepidum. In the former case, a firm correlation was established between the abrupt blue shift of the bacteriochlorophyll-a exciton absorption, a known indicator of the breakage of tertiary structure pigment-protein hydrogen bonds, and the quenching of tryptophan fluorescence, a supposed result of further protein solvation. No such effects were observed in the reference complex. While these data may be naively taken as supporting evidence of the governing role of hydration, the analysis of atomistic model structures of the complexes confirmed the critical part of the structure in the pressure-induced denaturation of the membrane proteins studied.

Pressure Unfolding of Proteins: New Insights into the Role of Bound Water.

High pressures can be detrimental for protein stability, resulting in unfolding and loss of function. This phenomenon occurs because the unfolding transition is accompanied by a decrease in volume, which is typically attributed to the elimination of cavities that are present within the native state as a result of packing defects. We present a novel computational approach that enables the study of pressure unfolding in atomistically detailed protein models in implicit solvent. We include the effect of pressure using a transfer free energy term that allows us to decouple the effect of protein residues and bound water molecules on the volume change upon unfolding. We discuss molecular dynamics simulations results using this protocol for two model proteins, Trp-cage and staphylococcal nuclease (SNase). We find that the volume reduction of bound water is the key energetic term that drives protein denaturation under the effect of pressure, for both Trp-cage and SNase. However, we note differences in unfolding mechanisms between the smaller Trp-cage and the larger SNase protein. Indeed, the unfolding of SNase, but not Trp-cage, is seen to be further accompanied by a reduction in the volume of internal cavities. Our results indicate that, for small peptides, like Trp-cage, pressure denaturation is driven by the increase in solvent accessibility upon unfolding, and the subsequent increase in the number of bound water molecules. For larger proteins, like SNase, the cavities within the native fold act as weak spots, determining the overall resistance to pressure denaturation. Our simulations display a striking agreement with the pressure-unfolding profile experimentally obtained for SNase and represent a promising approach for a computationally efficient and accurate exploration of pressure-induced denaturation of proteins.

The role of water in the impact of high pressure on the myrosinase activity and glucosinolate content in seedlings from Brussels sprouts

Abstract In this study, it was shown that the amount of available water was found to influence the high pressure processing (HPP) effect on both myrosinase activity and total glucosinolate concentration in Brussels sprouts seedlings. Brussels sprouts seedlings with different water content (wc = 4.8–89.4%) and water activity (aw = 0.17–0.97) were pressurized at selected pressures between 200 and 800 MPa (5 °C and 3 min), thereby affecting pressure-induced enzyme denaturation, molecular diffusion, and cell permeability differently. The myrosinase activity and intact glucosinolate content in the dry seedlings (wc Industrial relevance High pressure processing (HPP) is increasingly applied in the food industry. The treatment is acknowledged for the ability to give products longer shelf life concomitant with a high nutritional quality and fresh appearance. Upon HPP of cruciferous plants it is important to have a special focus on the glucosinolate-myrosinase system, since sub-optimal pre-treatment and/or processing parameters can affect the food quality negatively. The present study provides valuable results regarding the significance of water content and activity on the sensitivity of the myrosinase-glucosinolate system in Brussels sprouts seedlings towards HPP. Thus, providing a tool for designing different types of HPP products with respect to levels of active myrosinase and intact glucosinolates by adjustment of water content, water activity and HPP level.

Structural stability of human butyrylcholinesterase under high hydrostatic pressure

Human butyrylcholinesterase is a nonspecific enzyme of clinical, pharmacological and toxicological significance. Although the enzyme is relatively stable, its activity is affected by numerous factors, including pressure. In this work, hydrostatic pressure dependence of the intrinsic tryptophan fluorescence in native and salted human butyrylcholinesterase was studied up to the maximum pressure at ambient temperature of about 1200 MPa. A correlated large shift toward long wavelengths and broadening observed at pressures between 200 and 700 MPa was interpreted as due to high pressure-induced denaturation of the protein, leading to an enhanced exposure of tryptophan residues into polar solvent environment. This transient process in native butyrylcholinesterase presumably involves conformational changes of the enzyme at both tertiary and secondary structure levels. Pressure-induced mixing of emitting local indole electronic transitions with quenching charge transfer states likely describes the accompanying fluorescence quenching that reveals different course from spectral changes. All the pressure-induced changes turned irreversible after passing a mid-point pressure of about 400 ± 50 MPa. Addition of either 0.1 M ammonium sulphate (a kosmotropic salt) or 0.1 M lithium thiocyanate (a chaotropic salt) to native enzyme similarly destabilized its structure.

The opposing effect of urea and high pressure on the conformation of the protein β-hairpin: A molecular dynamics simulation study

It is a well-known fact that both concentrated aqueous urea solution (at ambient pressure) and high hydrostatic pressure, separately, cause the denaturation of proteins. But a combined effect of them i.e. the effect of high pressure on the protein conformation in concentrated aqueous urea solution is mostly unexplored except for one recent experimental study (see de Oliveira and Silva, 2015), which reported the counteracting ability of aqueous urea solution against pressure-induced denaturation of proteins. Thus, though urea causes denaturation of a protein at ambient pressure condition, remarkably, it protects protein's native conformation at high pressure. And, in the present article, the molecular insights into these rather unusual behaviors of urea molecules, at high hydrostatic pressure, towards the conformational stability of the protein β-hairpin are examined by employing classical molecular dynamics simulation. We notice that urea-protein-backbone interaction becomes less favorable as pressure is increased. Moreover, at high pressure, the presence of urea molecules makes water-protein interactions significantly less favorable when compared to that for low pressure. Thus, these findings lead us to propose that in binary urea solution, at high pressure, the retention of the protein's native conformation arises mainly due to (a) less exposure of protein-backbone for urea molecules and (b) significantly less favorable water-protein interactions.

Toward Extreme Biophysics: Deciphering the Infrared Response of Biomolecular Solutions at High Pressures.

Biophysics under extreme conditions is the fundamental platform for scrutinizing life in unusual habitats, such as those in the deep sea or continental subsurfaces, but also for putative extraterrestrial organisms. Therefore, an important thermodynamic variable to explore is pressure. It is shown that the combination of infrared spectroscopy with simulation is an exquisite approach for unraveling the intricate pressure response of the solvation pattern of TMAO in water, which is expected to be transferable to biomolecules in their native solvent. Pressure-enhanced hydrogen bonding was found for TMAO in water. TMAO is a molecule known to stabilize proteins against pressure-induced denaturation in deep-sea organisms.

Effect of high pressure and heat treatments on IgA immunoreactivity and lysozyme activity in human milk

Human milk from donor mothers is processed in human milk banks using low-temperature long-time pasteurization to ensure microbial safety. However, this treatment can degrade relevant bioactive components. High-pressure processing of food is a nonthermal treatment that exerts an antimicrobial effect maintaining the nutritional quality of foods. In this study, the effect of high pressure and heat treatments of human milk on denaturation of immunoglobulin A (IgA) and lysozyme activity was determined. Immunoreactive IgA was measured using a sandwich ELISA and lysozyme activity by a Micrococcus lysodeikticus turbidimetric assay. The retention kinetic of IgA in human milk treated by high pressure was studied. The experimental data obtained in the range of 350–650 MPa were well described by the Weibull model. The shape parameter (β) was not affected by the pressure, whereas the scale parameter (α) was affected by the pressure and its behavior was described by an Eyring-type equation. The estimated activation volume (ΔV a) was −25.67 ± 5.32 ml mol−1, and the constant reaction rate at the reference pressure (K p) was 0.022 ± 0.005 min−1. Kinetic parameters obtained allow estimating the pressure-induced denaturation of IgA on the basis of pressure and holding times. A substantial activation of lysozyme activity was obtained after treatment at pressures of 400, 450 and 500 MPa for 30 min. Treatment of human milk at 65 °C for 30 min maintained 43 % of IgA immunoreactivity, whereas lysozyme activity was not affected.

Influence of high-pressure processing on the physicochemical and the emulsifying properties of sarcoplasmic proteins from hake (Merluccius merluccius)

The sarcoplasmic proteins from hake (Merluccius merluccius) were subjected to high-pressure treatments (200, 400 and 600 MPa for 6 min at 20 °C). The physicochemical changes and the emulsifying capacity of sarcoplasmic proteins were evaluated. High-pressure processing increased the surface hydrophobicity and the reactive sulfhydryl group content of sarcoplasmic proteins above 200 MPa. Likewise, conformational changes were induced on the secondary structure of sarcoplasmic proteins at 400 MPa. High-pressure denaturation of sarcoplasmic proteins promoted their aggregation. The electrophoretic patterns showed that some sarcoplasmic proteins (78, 73, 63, 58, 37, 32 and 22 kDa) were the most pressure labile. However, the carbonyl content of sarcoplasmic proteins was not affected by high-pressure treatments. The pressure-induced denaturation affected the emulsifying properties of sarcoplasmic proteins, which was dependent on pressure. In contrast to high-pressure levels (400 and 600 MPa), sarcoplasmic proteins treated at 200 MPa produced emulsions with enhanced stability, greater consistency index, and shear-thinning behavior. These results suggested that high-pressure denaturation can affect the functional properties of sarcoplasmic proteins.

Mechanisms of pressure-mediated cell death and injury in Escherichia coli: from fundamentals to food applications.

High hydrostatic pressure is commercially applied to extend the shelf life of foods, and to improve food safety. Current applications operate at ambient temperature and 600 MPa or less. However, bacteria that may resist this pressure level include the pathogens Staphylococcus aureus and strains of Escherichia coli, including shiga-toxin producing E. coli. The resistance of E. coli to pressure is variable between strains and highly dependent on the food matrix. The targeted design of processes for the safe elimination of E. coli thus necessitates deeper insights into mechanisms of interaction and matrix-strain interactions. Cellular targets of high pressure treatment in E. coli include the barrier properties of the outer membrane, the integrity of the cytoplasmic membrane as well as the activity of membrane-bound enzymes, and the integrity of ribosomes. The pressure-induced denaturation of membrane bound enzymes results in generation of reactive oxygen species and subsequent cell death caused by oxidative stress. Remarkably, pressure resistance at the single cell level relates to the disposition of misfolded proteins in inclusion bodies. While the pressure resistance E. coli can be manipulated by over-expression or deletion of (stress) proteins, the mechanisms of pressure resistance in wild type strains is multi-factorial and not fully understood. This review aims to provide an overview on mechanisms of pressure-mediated cell death in E. coli, and the use of this information for optimization of high pressure processing of foods.

Simulation of Pressure-Induced and Temperature-Induced Denaturation of Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK) is a 415-residue protein composed of two domains of almost equal size. Under the heat-induced denaturation, folding and unfolding of PGK is a two-state process. However, folding and unfolding of PGK under pressure-induced denaturation,is a three-state process. How the temperature and pressure affect the denaturation of PGK is still unknown. We aim to provide a molecular explanation for thermal and pressure denaturation by using coarse-grained molecular simulations where the interactions of residues experience expulsion of water molecules.

Driving Forces in Pressure-Induced Protein Transitions.

The molecular mechanisms underlying pressure-induced protein denaturation can be analyzed based on the pressure-dependent differences in the apparent volume occupied by amino acids inside the protein and when exposed to water in an unfolded conformation. This chapter presents a volumetric analysis of the peptide group and the 20 naturally occurring amino acid side chains in the interior of the native state, the micelle-like interior of the pressure-induced denatured state, and in the unfolded conformation modeled by low-molecular analogs of proteins. The transfer of a peptide group from the protein interior to water becomes increasingly favorable as pressure increases. This observation classifies solvation of peptide groups as a major driving force in pressure-induced protein denaturation. Polar side chains do not appear to exhibit significant pressure-dependent changes in their preference for the protein interior or solvent. The transfer of nonpolar side chains from the protein interior to water becomes more unfavorable as pressure increases. An inference can be drawn that a sizeable population of nonpolar side chains remains buried inside a solvent-inaccessible core of the pressure-induced denatured state. At elevated pressures this core, owing to the absence of structural constraints, may become packed almost as tightly as the interior of the native state. The presence and partial disappearance of large intraglobular voids is another driving force facilitating pressure-induced protein denaturation. Volumetric data presented here have implications for the kinetics of protein folding and shed light on the nature of the folding transition state ensembles.

Pairwise association of neopentane as a function of hydrostatic pressure

It is shown that the contact-minimum configuration of two neopentane molecules is favoured on increasing hydrostatic pressure over the range 1–4000atm, at room temperature, in line with the methane results (Graziano, 2014). This is due to the decrease in water accessible surface area accompanying the association, that leads to a gain in configurational/translational entropy of water molecules, whose magnitude increases with hydrostatic pressure. Of course, the strengthening of pairwise hydrophobic interaction cannot shed light on the pressure-induced denaturation of globular proteins.

Kinetic parameters for high-pressure-induced denaturation of lactoferrin in human milk

The effect of high pressure and heat treatment of human milk on denaturation of lactoferrin was studied. The degree of protein denaturation was estimated using a sandwich enzyme-linked immunosorbent assay. Kinetic parameters for pressure-induced denaturation of lactoferrin were determined over the pressure range 300–650 MPa at 20 °C. Treatment at 300, 400, 500 and 600 MPa for 15 min denatured 9%, 23%, 34% and 48% of lactoferrin, respectively, whereas treatment at 65 °C for 30 min denatured 80%. Pressure-induced denaturation of lactoferrin followed a reaction order of n = 3. Values for the denaturation rate constant ranged from 1.9 × 104 s−1 at 300 MPa to 58.0 × 104 s−1 at 650 MPa. Heat-induced denaturation of lactoferrin involved protein aggregation by disulphide bonds; covalent interactions did not occur during pressure treatment. Results suggest that high-pressure processing is a potential alternative to thermal pasteurisation, giving greater retention of native lactoferrin.

Sorbitol counteracts high hydrostatic pressure-induced denaturation of inulin fructotransferase

Inulin fructotransferase (IFTase), a novel hydrolase for inulin, was exposed to high hydrostatic pressure (HHP) at 400 and 600MPa for 15min in the presence or absence of sorbitol. Sorbitol protected the enzyme against HHP-induced activity loss. The relative residual activity increased nearly 3.1- and 3.8-fold in the presence of 3mol/L sorbitol under 400MPa and 600MPa for 15min, respectively. Circular dichroism results indicated that the original chaotic unfolding conformation of the enzyme under HHP shifted toward more ordered and impact with 3mol/L sorbitol. Fluorescence and UV spectra results suggested that sorbitol prevented partially the unfolding of the enzyme and stabilized the conformation under high pressure. These results might be attributed to the binding of sorbitol on the surface of IFTase to rearrange and strengthen intra- and intermolecular hydrogen bonds.

Minima and Barriers on the Pressure-Temperature Free Energy Landscape of Phosphoglycerate Kinase

Proteins can exhibit strikingly different free energy landscapes depending on which thermodynamic variable is manipulated to study folding. We have investigated the relationship between temperature- and pressure-induced denaturation of yeast phosphoglycerate kinase (PGK). Thermodynamically, the pressure-temperature phase diagram of PGK seems standard: with a slight deviation from two-state behavior at low temperatures, a predicted elliptical shape of stability emerges. Kinetically, the behavior is much more complex: although a temperature perturbation of the energy landscape of PGK reveals a multitude of barriers, pressure-jump experiments spanning timescales from microsecond to hours showed only one predominant several-minute-long kinetic phase. These observations may indicate that under pressure, the smaller kinetic barriers of PGK are smoothed out by the large positive activation volume of the transition state.

Crucial Importance of Water Structure Modification on TrimethylamineN-Oxide Counteracting Effect at High Pressure

Penetration of water molecules into the protein interior under high hydrostatic pressure conditions, leading to protein structural transition, is a well-known phenomenon. The counteracting effect of a naturally occurring osmolyte, trimethylamine N-oxide (TMAO), against pressure-induced protein denaturation is also well-established. But, what is largely unknown is the mechanism by which TMAO counteracts this protein denaturation. So to provide a molecular level understanding of how TMAO protects proteins at high pressure, we report here molecular dynamics (MD) computer simulation results for aqueous solutions of N-methylacetamide (NMA) with different TMAO concentrations over a wide range of pressures relevant to protein denaturation. Hydration behavior of NMA is analyzed at different conditions chosen. It is observed that hydrostatic pressure leads to a significant compression of hydration shell of nonpolar groups and increases hydration number. The compression is relatively insignificant in the vicinity of hydrogen bonding sites. TMAO can prevent pressure-induced enhanced hydration of NMA molecules. Interaction of TMAO with NMA and the structural and dynamical properties of water (site-site radial distribution function, coordination number, hydrogen-bond number, and lifetime) are also investigated to find the origin of the counteracting action of TMAO. Our results confirm that TMAO and pressure have counteracting effects on the water structural and dynamical properties, giving an explanation as to how TMAO counteracts pressure-conferred denaturation of proteins.