Application of pressure perturbation calorimetry to lipid bilayers

Pressure perturbation calorimetry (PPC) is a new method that measures the heat consumed or released by a sample after a sudden pressure jump. The heat change can be used to derive the thermal volume expansion coefficient, alpha(V), as a function of temperature and, in the case of phase transitions, the volume change, DeltaV, occurring at the phase transition. Here we present the first report on the application of PPC to determine these quantities for lipid bilayers. We measure the volume changes of the pretransition and main transition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the thermal expansivity of the fluid phase of DMPC and of two unsaturated lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. The high sensitivity of PPC instrumentation gives accurate data for alpha(V) and DeltaV even upon the application of relatively low pressures of approximately 5 bar.     

Applications of pressure perturbation calorimetry in biophysical studies

Pressure perturbation calorimetry (PPC) is a relatively new and efficient technique, to study the volumetric properties of biomolecules in solution. In PPC, the coefficient of thermal expansion of the partial volume of the biomolecule is deduced from the heat consumed or produced after small isothermal pressure jumps (typically ± 5 bar). This strongly depends on the interaction of the biomolecule with the solvent or cosolvent as well as on its packing and internal dynamic properties. This technique, complemented by ultrasound velocity and densitometry, provides valuable insight into the basic thermodynamic properties of solvation and volume effects accompanying phase transitions and interactions of biomolecular systems. Here we review data on protein folding, ligand binding processes, and phospholipid phase transitions, together with discussion of interpretation and further significant applications.     

Phase transition kinetics of lipid bilayer membranes studied by time-resolved pressure perturbation calorimetry

The relaxation kinetics of aqueous lipid dispersions after a pressure jump (p-jump) was investigated using time-resolved pressure perturbation calorimetry (PPC). Analysis of the calorimetric response curves by deconvolution with the instrumental response function gives information about slow processes connected with the lipid phase transition. The lipid transition from the gel to the liquid-crystalline state was found to be a multi-step process with relaxation constants in the seconds range resolvable by time-resolved PPC and faster processes with relaxation times shorter than ca. 5 s that could not be resolved by the instrument. The faster processes comprise ca. 50% of the total heat uptake at the transition midpoint. This is the first calorimetric measurement showing the multi-step nature of the transition. The results are in good agreement with data obtained with other detection methods and with molecular modelling experiments describing the transition as a multi-step process with nucleation and growth steps.     

Hydration and packing effects on prion folding and beta-sheet conversion. High pressure spectroscopy and pressure perturbation calorimetry studies

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which undergoes aggregation and triggers the onset of transmissible spongiform encephalopathies. Here, we compare the stability against pressure and the thermomechanical properties of the alpha-helical and beta-sheet conformations of recombinant murine prion protein, designated as alpha-rPrP and beta-rPrP, respectively. High temperature induces aggregates and a large gain in intermolecular antiparallel beta-sheet (beta-rPrP), a conformation that shares structural similarity with PrP(Sc). alpha-rPrP is highly stable, and only pressures above 5 kilobars (1 kilobar = 100 MegaPascals) cause reversible denaturation, a process that leads to a random and turnrich conformation with concomitant loss of alpha-helix, as measured by Fourier transform infrared spectroscopy. In contrast, aggregates of beta-rPrP are very sensitive to pressure, undergoing transition into a dissociated species that differs from the denatured form derived from alpha-rPrP. The higher susceptibility to pressure of beta-rPrP can be explained by its less hydrated structure. Pressure perturbation calorimetry supports the view that the accessible surface area of alpha-rPrP is much higher than that of beta-rPrP, which explains the lower degree of hydration of beta-rPrP. Our findings shed new light on the mechanism of prion conversion and show how water plays a prominent role. Our results allow us to propose a volume and free energy diagram of the different species involved in the conversion and aggregation. The existence of different folded conformations as well as different denatured states of PrP may explain the elusive character of its conversion into a pathogenic form.     

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.     

Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes

The protection of mannosylglycerate, at 0.5 M concentration, against heat inactivation of the model enzyme lactate dehydrogenase (LDH) was compared to that exerted by other compatible solutes, namely, trehalose, ectoine, hydroxyectoine, di- myo-inositol phosphate, diglycerol phosphate, and mannosylglyceramide. Mannosylglycerate and hydroxyectoine were the best stabilizers of the enzyme and showed comparable protective effects. Diglycerol phosphate, trehalose, and mannosylglyceramide protected the enzyme to a lower extent. Ectoine conferred no protection, and di- myo-inositol phosphate had a strong destabilizing effect. The superior ability of mannosylglycerate to prevent LDH inactivation was accompanied by a higher efficiency in preventing LDH aggregation induced by heat stress. Moreover, mannosylglycerate induced an increase of 4.5 degrees C in the melting temperature of LDH, whereas the same molar concentration of trehalose caused an increase of only 2.2 degrees C. The effectiveness of mannosylglycerate in protecting LDH was also compared to that of other chemically related compounds: mannose, methyl-mannoside, potassium glycerate, glucosylglycerol, glycerol, and glucose. Mannosylglycerate conferred the highest protection, but glucosylglycerol and potassium glycerate were very efficient; glucose exerted a low degree of protection, glycerol and methyl-mannoside had no significant effect, and mannose caused destabilization. Mannosylglycerate was also a good thermoprotectant of glucose oxidase from Aspergillus niger, an enzyme with a net charge opposite to that of LDH under the working conditions. Given the superior performance of mannosylglycerate as a thermoprotectant of enzyme activity in vitro, it is conceivable that it also fulfills a protein thermoprotective function in vivo.     

Factors determining pressure perturbation calorimetry measurements: evidence for the formation of metastable states at lipid phase transitions

The factors that influence the application of pressure perturbation calorimetry in studying the volume change of the phase transition of lipids are discussed. These factors include a correction for the temperature-shift induced by perturbation, the kinetic irreversibility of the phase transition and the magnitude of the pressure perturbation. We take into account the fact that the dependence of the phase transition temperature on pressure will affect the temperature-shift induced by pressure. As a result, there is a discrepancy between the compression part of the cycle and the expansion. In addition, sequential cycles lead to a gradual loss in magnitude of the heat effect upon pressure perturbation. We suggest that these phenomena can be explained by the formation of a metastable glass-like state that converts to a stable phase at temperatures removed from the region of the phase transition.     

Determination of the catalytic activity of binuclear metallohydrolases using isothermal titration calorimetry

Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k _cat, K _m, and k _cat/K _m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate—the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected examples of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays.     

Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure

High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in solubilization yields of approximately 42%-58% from 1 mg/mL of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (approximately 70%-100%) to achieve refolding yields of approximately 55%-78% from IBs (1 mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.     

Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins

High‐density lipoproteins (HDLs) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic α‐helices that have high content (≥1/3) and distinct distribution of charged and apolar residues, adopt molten globule‐like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA‐I, shows relatively large volume contraction, ΔV_unf = ?0.33%, and an apparent reduction in thermal expansivity upon unfolding, Δα_unf ≤ 0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon α‐helical unfolding, which may result from disruption of multiple salt bridges. At 5°C, apoA‐I shows large thermal expansion coefficient, α(5°) = 15·10^?4 K^?1, that rapidly declines upon heating from 5 to 40°C, α(40°) ? α(5°) = ?4·10^?4 K^?1; apolipoprotein C‐I shows similar values of α(5°) and α(40°). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein–lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA‐I or apoC‐I, dimyristoyl phosphatidylcholine, and 0–20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Temperature stability of proteins: Analysis of irreversible denaturation using isothermal calorimetry

The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (～100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day^?1. Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.     

Rapid exploration of the folding topology of helical membrane proteins using paramagnetic perturbation

An understanding of the folding states of α-helical membrane proteins in detergent systems is important for functional and structural studies of these proteins. Here, we present a rapid and simple method for identification of the folding topology and assembly of transmembrane helices using paramagnetic perturbation in nuclear magnetic resonance spectroscopy. By monitoring the perturbation of signals from glycine residues located at specific sites, the folding topology and the assembly of transmembrane helices of membrane proteins were easily identified without time-consuming backbone assignment. This method is validated with Mistic (membrane-integrating sequence for translation of integral membrane protein constructs) of known structure as a reference protein. The folding topologies of two bacterial histidine kinase membrane proteins (SCO3062 and YbdK) were investigated by this method in dodecyl phosphocholine (DPC) micelles. Combing with analytical ultracentrifugation, we identified that the transmembrane domain of YbdK is present as a parallel dimer in DPC micelle. In contrast, the interaction of transmembrane domain of SCO3062 is not maintained in DPC micelle due to disruption of native structure of the periplasmic domain by DPC micelle.     

The effect of compatible solutes on proteins

Summary The ability of the compatible solute, proline, to affect the behavior of proteins has been examined in many different systems by many researches. In the present study of protein solvation, proline has been shown to prevent or diminish, in a concentration-dependent manner, the glutamine synthetase-precipitating ability of polyethylene glycol (PEG). The effects of PEG concentration and molecular weight are reduced by proline, and the interaction is strongly affected by pH.PEG causes precipitation of many proteins, and the ability of proline to reduce the precipitation of two non-enzymatic conjugated proteins, alfalfa mosaic virus and an³H-testosterone/antiserum complex, was also examined. Proline was effective in reducing the PEG-induced precipitation of both proteins. Virus precipitation by PEG and its alleviation by proline are influenced by pH. The increased virus-precipitating effect of PEG in the presence of salt (NaCl) is also alleviated by proline. The precipitation of the radioimmune complex by PEG is diminished by proline and by a mixture of free amino acids.These results indicate the generality of the three-way interaction between proline, protein and PEG. They may be of importance for extraction of proteins from biological systems and in studies of enzyme inactivation or protein denaturation in a cytoplasmic milieu. The results suggest that the protective effects of some amino acids are at least additive and are consistent with the conclusion that the compatible solutes protect protein-containing systems against the unfavorable consequences of dehydration and other stresses, by increasing the tendency of the system to maintain thestatus quo.     

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

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

The mechanism of cryoprotection of proteins by solutes

We have tested the capacity of 28 different compounds to protect lactate dehydrogenase from damage during freeze-thawing. These solutes come from very dissimilar chemical classes including sugars, polyols, amino acids, methylamines, and lyotropic salts. All the compounds tested, except NaCl, protected the enzyme, to varying degrees, from inactivation. The only characteristic that these compounds have in common, as a group, is that they have all been shown to be preferentially excluded from contact with the surface of proteins in aqueous solution. It has been demonstrated previously (via thermodynamic arguments) that this interaction of solutes with proteins leads to the stabilization of proteins in nonfrozen, aqueous systems. Conversely, those solutes, e.g., urea and guanidine HCl, that bind to proteins destabilize proteins in solution, and we have found that they also enhanced the inactivation of lactate dehydrogenase during freeze-thawing. Based on the results of our freeze-thawing experiments and a review of the theory of protein stabilization in nonfrozen, aqueous solution we propose that the cryoprotection afforded to isolated proteins by solutes can be accounted for by the fact that these solutes are preferentially excluded from contact with the protein's surface.     

Enzyme activities in concentrated solutions of glycinebetaine and other solutes

The activities of a number of enzymes in concentrated solutions of glycinebetaine and other solutes have been studied. Glycinebetaine, in contrast to electrolytes such as NaCl, was found to be noninhibitory up to 500 mM. This is compatible with the postulated role of glycinebetaine in cytoplasmic osmoregulation. Partial protection against NaCl inhibition was afforded by glycinebetaine in some cases. More detailed studies on glycinebetaine —NaCl-enzyme interactions were carried out using malate dehydrogenase (decarboxylating) from Hordeum vulgare.Abbreviations TES N-tris[hydroxymethyl]methyl-2-aminoethane sulphonic acid - MES 2[N-Morpholino]ethane sulphonic acid