Pressure Perturbation Calorimetry Research Articles

Interactions between small organic molecules and water measured using pressure perturbation calorimetry

Aqueous liquid mixtures play a critical role in many biological and chemical processes. Solutes including sugars, sugar alcohols, carboxylic acids, alcohols and acetone can affect the hydrogen-bonded structure of water and this can be measured using pressure perturbation calorimetry (PPC). In binary water–solute mixtures, Δ(∂CP/∂P)T is a measure of the structure of the water component. At low alcohol concentrations, negative Δ(∂CP/∂P)T values are consistent with clathrate-like water cages around the alkyl moieties. Conversely, when solutes hydrogen bond with water it interferes in the formation of “ice-like” water and is observable as a positive Δ(∂CP/∂P)T. The Δ(∂CP/∂P)T at increasing concentrations of ethanol, acetone and acetic acid in water displayed very different behaviors. Ethanol–water mixtures had three distinct concentration dependent phases; the first, with ethanol surrounded by water molecules, followed by the ethyl groups self-associating breaking the clathrate-like cages, and the ethanol–water network displacing all of the bulk water. Acetic acid–water mixtures display nonlinearity in Δ(∂CP/∂P)T versus acetic acid concentration consistent with acetic acid self-interaction which interferes with acetic acid capacity to disrupt water structure. Acetone-water mixtures display linearity in Δ(∂CP/∂P)T versus acetone concentration which is consistent with acetone’s inability to hydrogen bond with other acetone molecules. The lack of negative Δ(∂CP/∂P)T values in acetic acid-water and acetone-water mixtures suggests there is sufficient self-association between these solutes to prevent clathrate-like water cage formation. PPC can provide invaluable insight into the behavior of aqueous binary mixtures.

Eutectic Resolves Lysolipid Paradox in Thermoresponsive Liposomes.

A better molecular understanding of the temperature-triggered drug release from lysolipid-based thermosensitive liposomes (LTSLs) is needed to overcome the recent setbacks in developing this important drug delivery system. Enhanced drug release was previously rationalized in terms of detergent-like effects of the lysolipid monostearyl lysophosphatidylcholine (MSPC), stabilizing local membrane defects upon LTSL lipid melting. This is highly surprising and here referred to as the 'lysolipid paradox,' because detergents usually induce the opposite effect─they cause leakage upon freezing, not melting. Here, we aim at better answers to (i) why lysolipid does not compromise drug retention upon storage of LTSLs in the gel phase, (ii) how lysolipids can enhance drug release from LTSLs upon lipid melting, and (iii) why LTSLs typically anneal after some time so that not all drug gets released. To this end, we studied the phase transitions of mixtures of dipalmitoylphosphatidylcholine (DPPC) and MSPC by a combination of differential scanning and pressure perturbation calorimetry and identified the phase structures with small- and wide-angle X-ray scattering (SAXS and WAXS). The key result is that LTSLs, which contain the standard amount of 10 mol % MSPC, are at a eutectic point when they release their cargo upon melting at about 41 °C. The eutectic present below 41 °C consists of a MSPC-depleted gel phase as well as small domains of a hydrocarbon chain interdigitated gel phase containing some 30 mol % MSPC. In these interdigitated domains, the lysolipid is stored safely without compromising membrane integrity. At the eutectic temperature, both the MSPC-depleted bilayer and interdigitated MSPC-rich domains melt at once to fluid bilayers, respectively. Intact, fluid membranes tolerate much less MSPC than interdigitated domains─where the latter have melted, the high local MSPC content causes transient pores. These pores allow for fast drug release. However, these pores disappear, and the membrane seals again as the MSPC distributes more evenly over the membrane so that its local concentration decreases below the pore-stabilizing threshold. We provide a pseudobinary phase diagram of the DPPC-MSPC system and structural and volumetric data for the interdigitated phase.

Stretching the chains: the destabilizing impact of Cu2+ and Zn2+ ions on K48-linked diubiquitin.

Ubiquitin signalling and metal homeostasis play key roles in controlling several physiological cellular activities, including protein trafficking and degradation. While some relationships between these two biochemical pathways have started to surface, our knowledge of their interplay remains limited. Here, we employ a variety of techniques, such as circular dichroism, differential scanning calorimetry, pressure perturbation calorimetry, fluorescence emission, SDS-PAGE, and small-angle X-ray scattering (SAXS) to evaluate the impact of Cu2+ and Zn2+ ions on the structure and stability of K48 linked diubiquitin (K48-Ub2), a simple model for polyubiquitin chains. The SAXS analysis results show that the structure of the metal-free protein is similar to that observed when the protein is bound to the E2 conjugating enzyme, lending support to the idea that the structure of unanchored K48-linked ubiquitin chains is sufficient for identification by conjugating enzymes without the need for an induced fit mechanism. Our results indicate that K48-Ub2 can coordinate up to four metal ions with both copper and zinc ions inducing slight changes to the secondary structure of the protein. However, we noted significant distinctions in their impacts on protein stability and overall architecture. Specifically, Cu2+ ions resulted in a destabilization of the protein structure, which facilitated the formation of dimer aggregates. Next, we observed a shift in the conformational dynamics of K48-Ub2 toward less compact and more flexible states upon metal ion binding, with Zn2+ inducing a more significant effect than Cu2+ ions. Our structural modelling study demonstrates that both metal ions induced perturbations in the K48-Ub2 structure, leading to the separation of the two monomers thus inhibiting interactions with E2 enzymes. In conclusion, the findings from this study enhance our comprehension of the mechanisms underlying Ub chains recognition. Moreover, they strengthen the notion that drug discovery initiatives aimed at targeting metal-mediated disruptions in Ub signaling hold great potential for treating a wide range of diseases that stem from abnormal protein accumulation.

The volume changes of unfolding of dsDNA

High hydrostatic pressure can have profound effects on the stability of biomacromolecules. The magnitude and direction (stabilizing or destabilizing) of this effect is defined by the volume changes in the system, ΔV. Positive volume changes will stabilize the starting native state, whereas negative volume changes will lead to the stabilization of the final unfolded state. For the DNA double helix, experimental data suggested that when the thermostability of dsDNA is below 50°C, increase in hydrostatic pressure will lead to destabilization; i.e., helix-to-coil transition has negative ΔV. In contrast, the dsDNA sequences with the thermostability above 50°C showed positive ΔV values and were stabilized by hydrostatic pressure. In order to get insight into this switch in the response of dsDNA to hydrostatic pressure as a function of temperature, first we further validated this trend using experimental measurements of ΔV for 10 different dsDNA sequences using pressure perturbation calorimetry. We also developed a computational protocol to calculate the expected volume changes of dsDNA unfolding, which was benchmarked against the experimental set of 50 ΔV values that included, in addition to our data, the values from the literature. Computation predicts well the experimental values of ΔV. Such agreement between computation and experiment lends credibility to the computation protocol and provides molecular level rational for the observed temperature dependence of ΔV that can be traced to the hydration. Difference in the ΔV value for A/T versus G/C basepairs is also discussed.

Cosolvent and Crowding Effects on the Temperature- and Pressure-Dependent Dissociation Process of the α/β-Tubulin Heterodimer.

Tubulin is one of the main components of the cytoskeleton of eukaryotic cells. The formation of microtubules depends strongly on environmental and solution conditions, and has been found to be among the most pressure sensitive processes in vivo. We explored the effects of different types of cosolvents, such as trimethylamine-N-oxide (TMAO), sucrose and urea, and crowding agents to mimic cell-like conditions, on the temperature and pressure stability of the building block of microtubules, i. e. the α/β-tubulin heterodimer. To this end, fluorescence and FTIR spectroscopy, differential scanning and pressure perturbation calorimetry as well as fluorescence anisotropy and correlation spectroscopies were applied. The pressure and temperature of dissociation of α/β-tubulin as well as the underlying thermodynamic parameters upon dissociation, such as volume and enthalpy changes, have been determined for the different solution conditions. The temperature and pressure of dissociation of the α/β-tubulin heterodimer and hence its stability increases dramatically in the presence of TMAO and the nanocrowder sucrose. We show that by adjusting the levels of compatible cosolutes and crowders, cells are able to withstand deteriorating effects of pressure even up to the kbar-range.

Effect of ectoine, hydroxyectoine and β-hydroxybutyrate on the temperature and pressure stability of phospholipid bilayer membranes of different complexity.

Previous research has shown that ectoines fluidize lipid monolayers by increasing the liquid expanded region in DPPC monolayers and also decreasing the line tension responsible for the phase morphology. Here, we explored possible effects of the compatible osmolytes ectoine, hydroxyectoine and β-hydroxybutyrate on lipid bilayer membranes, including effects of temperature and pressure. The effect of the protective osmolytes on the phase transition of DPPC bilayers was investigated by fluorescence spectroscopy, differential scanning calorimetry and pressure perturbation calorimetry. A slight change of the phase behavior was observed, which resulted in a stabilization of the gel phase, which may be caused by an alteration of the hydration properties at the lipid interface and H-bond and electrostatic interactions in the headgroup region. We then explored the cosolvents' effects on giant unilamellar vesicles (GUVs) formed by lipid mixtures exhibiting phase separation into liquid-ordered (lo) and liquid-disordered (ld) domains using BODIPY-PC and the DiI18 dye as labels. The presence of both, ectoine and hydroxyectoine showed significant effects on the lateral organization increasing the fluid domains. Moreover, we observed a considerable increase in the adhesion behavior of small vesicles onto GUV surfaces. Diffusion studies by fluorescence recovery after photobleaching experiments on POPC giant vesicles quantitatively showed a hydroxyectoine-induced increase of the diffusion coefficient values, clearly demonstrating an increase in the lateral mobility of lipid within the bilayer membrane. This study provides clear evidence for the fluidizing effect of the compatible solutes on bilayer lipid membranes. A marked effect, however, was only detected if phase separated domains exist.

Protocols of IATC, DSC, and PPC: The Multistate Structural Transition of Cytochrome c.

The recent development of high-precision calorimeters allows us to monitor the structural transition of biomolecules by calorimetry and thereby characterize the thermodynamic property changes accompanying three-dimensional structure changes. We developed isothermal acid-titration calorimetry (IATC) to evaluate the pH dependence of protein enthalpy. Using the double deconvolution method with precise differential scanning calorimetry (DSC), we revealed that the MG state is an equilibrium intermediate state of the reversible thermal three-state transition of the protein, and we successfully determined its volumetric properties by pressure perturbation calorimetry (PPC). Our findings underscore the importance of a precise calorimetry and analysis model for protein research.

Pressure Perturbation Calorimetry: Application to Lipid Membranes

Pressure Perturbation Calorimetry: Application to Lipid Membranes

Small Angle Neutron Scattering Studies of R67 Dihydrofolate Reductase, a Tetrameric Protein with Intrinsically Disordered N-Termini.

R67 dihydrofolate reductase (DHFR) is a homotetramer with a single active site pore and no sequence or structural homology with chromosomal DHFRs. The R67 enzyme provides resistance to trimethoprim, an active site-directed inhibitor of Escherichia coli DHFR. Sixteen to twenty N-terminal amino acids are intrinsically disordered in the R67 dimer crystal structure. Chymotrypsin cleavage of 16 N-terminal residues results in an active enzyme with a decreased stability. The space sampled by the disordered N-termini of R67 DHFR was investigated using small angle neutron scattering. From a combined analysis using molecular dynamics and the program SASSIE (http://www.smallangles.net/sassie/SASSIE_HOME.html), the apoenzyme displays a radius of gyration (Rg) of 21.46 ± 0.50 Å. Addition of glycine betaine, an osmolyte, does not result in folding of the termini as the Rg increases slightly to 22.78 ± 0.87 Å. SASSIE fits of the latter SANS data indicate that the disordered N-termini sample larger regions of space and remain disordered, suggesting they might function as entropic bristles. Pressure perturbation calorimetry also indicated that the volume of R67 DHFR increases upon addition of 10% betaine and decreased at 20% betaine because of the dehydration of the protein. Studies of the hydration of full-length R67 DHFR in the presence of the osmolytes betaine and dimethyl sulfoxide find around 1250 water molecules hydrating the protein. Similar studies with truncated R67 DHFR yield around 400 water molecules hydrating the protein in the presence of betaine. The difference of ∼900 waters indicates the N-termini are well-hydrated.

Putting the Piezolyte Hypothesis under Pressure

A group of small molecules that stabilize proteins against high hydrostatic pressure has been classified as piezolytes, a subset of stabilizing cosolutes. This distinction would imply that piezolytes counteract the effects of high hydrostatic pressure through effects on the volumetric properties of the protein. The purpose of this study was to determine if cosolutes proposed to be piezolytes have an effect on the volumetric properties of proteins through direct experimental measurements of volume changes upon unfolding of model proteins lysozyme and ribonuclease A, in solutions containing varying cosolute concentrations. Solutions containing the proposed piezolytes glutamate, sarcosine, and betaine were used, as well as solutions containing the denaturants guanidinium hydrochloride and urea. Changes in thermostability were monitored using differential scanning calorimetry whereas changes in volume were monitored using pressure perturbation calorimetry. Our findings indicate that increasing stabilizing cosolute concentration increases the stability and transition temperature of the protein, but does not change the temperature dependence of volume changes upon unfolding. The results suggest that the pressure stability of a protein in solution is not directly affected by the presence of these proposed piezolytes, and so they cannot be granted this distinction.

Thermodynamics of the Thermal Denaturation of Acid Molten Globule State of Cytochrome c Indicate a Reversible High-Temperature Oligomerization Process.

In this study, we performed differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) analysis of the thermal transition of cytochrome c from an acidic molten globule (MG) state with the protein concentrations of 0.5-18.2 mg/mL. DSC profiles were highly reversible and showed clear protein-concentration dependence, indicating that reversible oligomerization occurred accompanying the thermal transition from the MG state. The DSC and PPC data required at least a six-state model (MG1 ⇄ MG2 ⇄ D ⇄ 1/2 I2 ⇄ 1/3 I3 ⇄ 1/4 I4) including three new oligomeric states: dimer (I2), trimer (I3), and tetramer (I4) in addition to the three monomeric states previously characterized. Dynamic light scattering confirmed the oligomerization during the thermal transition. The partial specific volumes of these oligomeric states were found to be smaller than those of the monomeric states, MG2 and D, indicating dehydration of hydrophobic surface or hydration of released anions may occur with the reversible oligomerization.

Analysis of Mesoscopic Structured 2-Propanol/Water Mixtures Using Pressure Perturbation Calorimetry and Molecular Dynamic Simulation

In this paper we demonstrate the application of pressure perturbation calorimetry (PPC) to the characterization of 2-propanol/water mixtures. PPC of different 2-propanol/water mixtures provides two useful measurements: (i) the change in heat (ΔQ); and (ii) the left[ {delta bar{C}_{p} /delta p} right]_{T} value. The results demonstrate that the ΔQ values of the mixtures deviate from that expected for a random mixture, with a maximum at ~20–25 mol% 2-propanol. This coincides with the concentration at which molecular dynamics (MD) simulations show a maximum deviation from random distribution, and also the point at which alcohol–alcohol hydrogen bonds become dominant over alcohol–water hydrogen bonds. Furthermore, the left[ {delta bar{C}_{p} /delta p} right]_{T} value showed transitions at 2.5 mol% 2-propanol and at approximately 14 mol% 2-propanol. Below 2.5 mol% 2-propanol the values of left[ {delta bar{C}_{p} /delta p} right]_{T} are negative; this is indicative of the presence of isolated 2-propanol molecules surrounded by water molecules. Above 2.5 mol% 2-propanol left[ {delta bar{C}_{p} /delta p} right]_{T} rises, reaching a maximum at ~14 mol% corresponding to a point where mixed alcohol–water networks are thought to dominate. The values and trends identified by PPC show excellent agreement not only with those obtained from MD simulations but also with results in the literature derived using viscometry, THz spectroscopy, NMR and neutron diffraction.

IATC, DSC, and PPC Analysis of Reversible and Multistate Structural Transition of Cytochrome c.

Development of precise calorimeters has enabled us to monitor the structural transition of biomolecules by calorimetry to characterize the thermodynamic property changes accompanying three-dimensional structure change. We developed isothermal acid-titration calorimetry to evaluate the pH dependence of protein enthalpy, and demonstrated the thermodynamic transition between the native and molten globule (MG) states of cytochrome c with very small enthalpy change (~20 kJ/mol) by this method. The double deconvolution method with precise differential scanning calorimetry has revealed the MG state as an equilibrium intermediate state of the reversible thermal transition of the protein, and pressure perturbation calorimetry has succeeded in determining its volumetric properties. These examples strongly indicate the importance of a precise calorimetry and analysis model in the field of protein research.

Applications of pressure perturbation calorimetry to study factors contributing to the volume changes upon protein unfolding

BackgroundPressure perturbation calorimetry (PPC) is a biophysical method that allows direct determination of the volume changes upon conformational transitions in macromolecules. Scope of this reviewThis review provides novel details of the use of PPC to analyze unfolding transitions in proteins. The emphasis is made on the data analysis as well as on the validation of different structural factors that define the volume changes upon unfolding. Four case studies are presented that show the application of these concepts to various protein systems. Major conclusionsThe major conclusions are:1.Knowledge of the thermodynamic parameters for heat induced unfolding facilitates the analysis of the PPC profiles.2.The changes in the thermal expansion coefficient upon unfolding appear to be temperature dependent.3.Substitutions on the protein surface have negligible effects on the volume changes upon protein unfolding.4.Structural plasticity of proteins defines the position dependent effect of amino acid substitutions of the residues buried in the native state.5.Small proteins have positive volume changes upon unfolding which suggests difference in balance between the cavity/void volume in the native state and the hydration volume changes upon unfolding as compared to the large proteins that have negative volume changes. General significanceThe information provided here gives a better understanding and deeper insight into the role played by various factors in defining the volume changes upon protein unfolding. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.

Thermal expansivities of peptides, polypeptides and proteins as measured by pressure perturbation calorimetry

The main goal of this work was to provide direct experimental evidence that the expansivity of peptides, polypeptides and proteins as measured by pressure perturbation calorimetry (PPC), can serve as a proxy to characterize relative compactness of proteins, especially the denatured state ensemble. This is very important as currently only small angle X-ray scattering (SAXS), intrinsic viscosity and, to a lesser degree, fluorescence resonance transfer (FRET) experiments are capable of reporting on the compactness of denatured state ensembles. We combined the expansivity measurements with other biophysical methods (far-UV circular dichroism spectroscopy, differential scanning calorimetry, and small angle X-ray scattering). Three case studies of the effects of conformational changes on the expansivity of polypeptides in solution are presented. We have shown that expansivity appears to be insensitive to the helix–coil transition, and appears to reflect the changes in hydration of the side-chains. We also observed that the expansivity is sensitive to the global conformation of the polypeptide chain and thus can be potentially used to probe hydration of different collapsed states of denatured or even intrinsically disordered proteins.

A study of the relationship between water and anions of the Hofmeister series using pressure perturbation calorimetry.

Pressure perturbation calorimetry (PPC) was used to study the relationship between water and sodium salts with a range of different anions. At temperatures around 25 °C the heat on pressurisation (ΔQ) from 1 to 5 bar was negative for all solutions relative to pure water. The raw data showed that as the temperature rose, the gradient was positive relative to pure water and the transition temperature where ΔQ was zero was related to anion surface charge density and was more pronounced for the low-charge density anions. A three component model was developed comprising bulk water, the hydration layer and the solute to calculate the molar expansivity of the hydration layer around the ions in solution. The calculated molar expansivities of water in the hydration layer around the ions were consistently less than pure water. ΔQ at different disodium hydrogen phosphate concentrations showed that the change in molar enthalpy relative to pure water was not linear even as it approached infinite dilution suggesting that while hydration layers can be allocated to the water around ions this does not rule out interactions between water and ions extending beyond the immediate hydration layer.

圧力摂動熱量法によるリン脂質二分子膜の体積挙動および緩和挙動の定量的観測

圧力摂動熱量法によるリン脂質二分子膜の体積挙動および緩和挙動の定量的観測

Exploring the Stability Limits of Actin and its Suprastructures

Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. G-actin is able to assemble into highly organized, supramolecular cellular structures, filamentous (F) and bundled (B) actin. To evaluate the structure and establish the stability of G-, F- and B- actin over a wide range of temperatures and pressures, FTIR spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle X-ray scattering, laser confocal scanning and transmission electron microscopy was used. On the one hand, our analysis was designed to provide new insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including conditions encountered in extreme environments. On the other hand, our data help understand the limited pressure stability of actin self-assembly observed in vivo. G-actin is not only the least temperature-stable, but also the least pressure-stable actin species. At abyssal conditions, where temperatures as low as 1-4°C and pressures up to 1 kbar are reached, G-actin is hardly stable anymore. The supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth, however. Beyond ∼3-4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between about 1 and 2 kbar, some disordering of actin assemblies commences already, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range.

Biophysical Analysis of a Successful Protocol to Reconstitute Tetramers of the M2 Muscarinic Receptor

The human G protein-coupled receptor M2 muscarinic receptor has been functionally reconstituted in its tetrameric state into mixed lipid bilayers (Redka et al. 2013). This is achieved by first solubilizing the receptor in mixed detergent micelles composed of digitonin and sodium cholate, then reconstituting it into vesicles composed of phosphatidylcholine, phosphatidylserine and cholesterol, followed by detergent removal. To understand how the individual detergent and lipid components used in this empirical protocol contribute to the stability and activity of the receptor, we used isothermal titration calorimetry (ITC) to study the self-assembly of the mixed surfactant system; differential scanning calorimetry and pressure perturbation calorimetry to probe the phase behavior of the membrane; ITC, fluorescence (time-resolved) leakage assays and dynamic light scattering to characterize detergent-lipid interactions. The results suggest ideal mixing between digitonin and sodium cholate in the formation of mixed micelles that stabilize the receptor. Differences in membrane-partitioning behavior between the two detergents and the presence of a significant fraction of gel phase at the temperature used in the protocol contribute to non-equilibration of the detergents in the bilayer. The insights gained from this biophysical approach will aid in the mechanistic selection of detergents and conditions that influence function, oligomeric state, orientation, and accessibility of membrane proteins in future studies.

Exploring the Stability Limits of Actin and Its Suprastructures

Actin is the main component of the microfilament system in eukaryotic cells and can be found in distinct morphological states. Global (G)-actin is able to assemble into highly organized, supramolecular cellular structures known as filamentous (F)-actin and bundled (B)-actin. To evaluate the structure and stability of G-, F-, and B-actin over a wide range of temperatures and pressures, we used Fourier transform infrared spectroscopy in combination with differential scanning and pressure perturbation calorimetry, small-angle x-ray scattering, laser confocal scanning microscopy, and transmission electron microscopy. Our analysis was designed to provide new (to our knowledge) insights into the stabilizing forces of actin self-assembly and to reveal the stability of the actin polymorphs, including in conditions encountered in extreme environments. In addition, we sought to explain the limited pressure stability of actin self-assembly observed in vivo. G-actin is not only the least temperature-stable but also the least pressure-stable actin species. Under abyssal conditions, where temperatures as low as 1–4°C and pressures up to 1 kbar are reached, G-actin is hardly stable. However, the supramolecular assemblies of actin are stable enough to withstand the extreme conditions usually encountered on Earth. Beyond ∼3–4 kbar, filamentous structures disassemble, and beyond ∼4 kbar, complete dissociation of F-actin structures is observed. Between ∼1 and 2 kbar, some disordering of actin assemblies commences, in agreement with in vivo observations. The limited pressure stability of the monomeric building block seems to be responsible for the suppression of actin assembly in the kbar pressure range.