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BBA - Proteins and Proteomics (v.1764, #3)

Editorial Board (pp. ii).

Proteins under high pressure by Lazlo Smeller; Hannelore Roemich; Reinhard Lange (pp. 329-330).

Conformational fluctuations of proteins revealed by variable pressure NMR by Hua Li; Kazuyuki Akasaka (pp. 331-345).

With the high-resolution variable-pressure NMR spectroscopy, one can study conformational fluctuations of proteins in a much wider conformational space than hitherto explored by NMR and other spectroscopic techniques. This is because a protein in solution generally exists as a dynamic mixture of conformers mutually differing in partial molar volume, and pressure can select the population of a conformer according to its relative volume. In this review, we describe how variable-pressure NMR can be used to probe conformational fluctuations of proteins in a wide conformational space from the folded to the fully unfolded structures, with actual examples. Furthermore, the newly emerging technique “NMR snapshots�? expresses amply fluctuating protein structures as changes in atomic coordinates. Finally, the concept of conformational fluctuation is extended to include intermolecular association leading to amyloidosis.

Keywords: Variable-pressure NMR; Protein conformational fluctuation; Higher energy conformer; Compressibility; Volume theorem of protein; NMR snapshot

Protein stability and dynamics in the pressure–temperature plane by Filip Meersman; László Smeller; Karel Heremans (pp. 346-354).

The pressure–temperature stability diagram of proteins and the underlying assumptions of the elliptical shape of the diagram are discussed. Possible extensions, such as aggregation and fibril formation, are considered. An important experimental observation is the extreme pressure stability of the mature fibrils. Molecular origins of the diagram in terms of models of the partial molar volume of a protein focus on cavities and hydration. Changes in thermal expansivity, compressibility and heat capacity in terms of fluctuations of the enthalpy and volume change of the unfolding should also focus on these parameters. It is argued that the study of water-soluble polymers might further our understanding of the stability diagram. Whereas the role of water in protein behaviour is unquestioned, the role of cavities is less clear.

Keywords: Abbreviations; p; T pressure–temperature; PPC; pressure perturbation calorimetry; FTIR; Fourier transform infrared; DSC; differential scanning calorimetryPressure–temperature phase diagram; Hydration; Cavity; Unfolding; Aggregation; Protein dynamics

Understanding high pressure stability of helical conformation of oligopeptides and helix bundle protein by Takahiro Takekiyo; Takashi Imai; Minoru Kato; Yoshihiro Taniguchi (pp. 355-363).

The pressure effect on conformational equilibria of simple organic compounds and the pressure denaturation of proteins have been well investigated by using vibrational spectroscopy. However, there was no systematic investigation of the pressure effect on conformational equilibria of oligopeptides, which are located between the simple organic compounds and proteins. Here, we review the recent vibrational spectroscopic and theoretical studies of the pressure effect on conformational equilibria of model oligopeptides and helix bundle protein in aqueous solution.

Keywords: Abbreviations; Ac; acetyl(CH; ₃; CO-); Ac-Ala-NHMe; N; -acetyl-; l; -alanine-; N; ′-methylamide; Ac-Gly-NHMe; N; -acetyl-; l; -glycine-; N; ′-methylamide; AKA, peptide; Ac-(AAAAK); ₄; -AAAAY-NH; ₂; AK16; YGAAKAAAAKAAAAKA-NH; ₂; CD; circular dichroism; D; denatured state; FT-IR; Fourier transform-infrared; HNC; hypernetted chain; KB; Kirkwood-Buff; NMA; N; -methylacetamide; N; native state; NMR; nuclear magnetic resonance; PLGA; poly-; l; -glutamic acid; PMV; partial molar volume; RISM; reference interaction site model; TFA; trifluoroacetic acidOligopeptide; de novo design protein; Pressure effect; Partial molar volume; Vibrational spectroscopy; RISM theory

Synergetic effects of pressure and chemical denaturant on protein unfolding: Stability of a serine-type carboxyl protease, kumamolisin by Yasunori Fujimoto; Hidekazu Ikeuchi; Tomoko Tada; Hiroshi Oyama; Kohei Oda; Shigeru Kunugi (pp. 364-371).

Kumamolisin, a serine carboxyl proteinase, is very stable and hardly denatured by single perturbation of a chemical denaturant (urea), pressure (<500 MPa) or temperature (<65 °C). In order to investigate the cooperative effects of these three denaturing agents, DSC, CD, intrinsic fluorescence, and fourth derivative UV absorbance were measured under various conditions. By application of pressure to kumamolisin in 8 M urea solution, substantial red-shift in the center of fluorescence emission spectral mass was observed, and the corresponding blue-shift was observed for two major peaks in fourth derivative UV absorbance, under the similar urea-containing conditions. The denaturation curves were analyzed on the basis of a simple two-state model in order to obtain thermodynamic parameters (Δ V, Δ G, and m values), and the combined effects of denaturing agents are discussed, with the special interest in the large cavity and neighboring Trp residue in kumamolisin.

Keywords: Serine carboxyl protease; Pressure; Urea; Denaturation; Cavity; Protein Unfolding; Kumamolisin; Sedolisin

sHSPs under temperature and pressure: The opposite behaviour of lens alpha-crystallins and yeast HSP26 by Fériel Skouri-Panet; Sophie Quevillon-Cheruel; Magalie Michiel; Annette Tardieu; Stéphanie Finet (pp. 372-383).

Small angle X-ray scattering was used to follow the temperature and pressure induced structural transitions of polydisperse native calf lens alpha-crystallins and recombinant human alphaB-crystallins and of monodisperse yeast HSP26. The alpha-crystallins were known to increase in size with increasing temperature, whereas HSP26 partially dissociates into dimers. SAXS intensity curves demonstrated that the average 40-mer calf alpha-crystallin converted into 80-mer in a narrow temperature range, from 60 to 69 °C, whereas the average 30-mer alphaB-crystallin was continuously transformed into 60-mer at lower temperature, from 40 to 60 °C. These temperature-induced transitions were irreversible. Similar transitions, yet reversible, could be induced with pressure in the 100 to 300 MPa pressure range. Moreover, temperature and pressure could be combined to lower the transition temperatures. On the other hand, SAXS curves recorded during pressure scans from 0.1 to 200 MPa with monodisperse 24-mer HSP26 revealed dissociation of the 24-mer into dimers. This dissociation was complete and reversible. Whatever the sHSP, a decrease of partial specific volume was found to be associated with the pressure induced quaternary structure transitions, in agreement with the hypothesis that such transitions represent a first step on the protein denaturation pathway.

Keywords: Small angle X-ray scattering; Small heat shock protein; Alpha-crystallin; HSP26; Temperature and pressure induced transition

High-Pressure Macromolecular Crystallography (HPMX): Status and prospects by Roger Fourme; Eric Girard; Richard Kahn; Anne-Claire Dhaussy; Mohamed Mezouar; Nathalie Colloc'h; Isabella Ascone (pp. 384-390).

Recent technical developments, achievements and prospects of high-pressure (HP) macromolecular crystallography (MX) are reviewed. Technical difficulties associated with this technique have been essentially solved by combining synchrotron radiation of ultra-short wavelength, large-aperture diamond anvil cells and new sample-mounting techniques. The quality of diffraction data collected at HP can now meet standards of conventional MX.The exploitation of the potential of the combination of X-ray diffraction and high-pressure perturbation is progressing well. The ability of pressure to shift the population distribution of conformers in solution, which is exploited in particular by NMR, can also be used in the crystalline state with specific advantages. HPMX has indeed bright prospects, in particular to elucidate the structure of higher-energy conformers that are often of high biological significance.Furthermore, HPMX may be of interest for conventional crystallographic studies, as pressure is a fairly general tool to improve order in pre-existing crystals with minimal perturbation of the native structure.

Keywords: Hydrostatic pressure; Protein crystallography

High pressure macromolecular crystallography: The 140-MPa crystal structure at 2.3 Å resolution of urate oxidase, a 135-kDa tetrameric assembly by Nathalie Colloc'h; Eric Girard; Anne-Claire Dhaussy; Richard Kahn; Isabella Ascone; Mohamed Mezouar; Roger Fourme (pp. 391-397).

We report the three-dimensional structure determined by high-pressure macromolecular crystallography (HPMX) of a 135-kDa homo-tetrameric enzyme, urate oxidase from Aspergillus flavus complexed with its potent inhibitor 8-azaxanthin. Urate oxidase crystals are quite sensitive to pressure, as three-dimensional order is lost at about 180 MPa. A highly complete 2.3 Å resolution data set was collected at 140 MPa, close to the critical pressure. Crystal structures at atmospheric pressure and at high pressure were refined in the orthorhombic space group I222 with final crystallographic R factors 14.1% and 16.1%, respectively. The effect of pressure on temperature factors, ordered water molecules, hydrogen bond lengths, contacts, buried surface areas as well as cavity volume was investigated. Results suggest that the onset of disruption of the tetrameric assembly by pressure has been captured in the crystalline state.

Keywords: Hydrostatic pressure; Protein crystallography; Urate oxidase; Oligomeric association; Folding; Protein destabilization

The effects of temperature, pressure and peptide incorporation on ternary model raft mixtures—A Laurdan fluorescence spectroscopy study by Nagarajan Periasamy; Roland Winter (pp. 398-404).

Recently, an increasing evidence accumulated for the existence of lipid microdomains, called lipid rafts, in cell membranes, which may play an important role in many important membrane-associated biological processes. Suitable model systems for studying biophysical properties of lipid rafts are lipid vesicles composed of three-component lipid mixtures, such as POPC/SM/cholesterol, which exhibit a rich phase diagram, including raft-like liquid-ordered/liquid-disordered phase coexistence regions. We explored the temperature, pressure and concentration-dependent phase behavior of such canonical model raft mixtures using the Laurdan fluorescence spectroscopic technique. Hydrostatic pressure has not only been used as a physical parameter for studying the stability and energetics of these systems, but also because high pressure is an important feature of certain natural membrane environments. We show that the liquid-disordered/liquid-ordered phase coexistence regions of POPC/SM/cholesterol model raft mixtures extends over a very wide temperature range of about 50 °C. Upon pressurization, an overall ordered membrane state is reached at pressures of ∼1000 bar at 20 °C, and of ∼2000 bar at 40 °C. Incorporation of 5 mol% gramicidin as a model ion channel slightly increases the overall order parameter profile in the l_o+l_d two-phase coexistence region, probably by selectively partitioning into l_d domains, does not change the overall phase behavior, however. This behavior is in contrast to the effect of the peptide incorporation into simple, one-component phospholipid bilayer systems.

Keywords: Abbreviations; GP; general polarization; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-phosphatidylcholine; SM; sphingomyelin; Chol; cholesterol; GD; gramicidin D; l; _d; liquid-disordered; l; _o; liquid-ordered; s; _o; solid-ordered; TMA-DPH; 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene; Laurdan; 6-dodecanoyl-2-dimethyl-aminonapthaleneHigh pressure; Model biomembrane; Phosholipid; Fluorescence spectrocopy; Peptide

Light scattering studies of proteins under compression by Ewa Banachowicz (pp. 405-413).

The scattering techniques are very convenient and effective in investigation of the shape, size and interactions of biological molecules close to their natural states in solution. However, it seems that from among a wide spectrum of scattering techniques, the light scattering ones have been relatively rarely used for the study of proteins under elevated hydrostatic pressure. This paper gives a brief description of the well developed possibilities of this technique for potential applications in the study of dissociation, aggregation and structural changes in proteins under compression. A short review of the already known applications is also given. Finally, the high-pressure dynamic light scattering results obtained by author on the lysozyme solution are shown and discussed.

Keywords: Light-scattering; Diffusion coefficient; Dissociation; High hydrostatic pressure; Protein

Influence of pressure on structure and dynamics of bovine pancreatic trypsin inhibitor (BPTI): Small angle and quasi-elastic neutron scattering studies by M.-S. Appavou; G. Gibrat; M.-C. Bellissent-Funel (pp. 414-423).

We have studied the influence of pressure on structure and dynamics of a small protein belonging to the enzymatic catalysis: the bovine pancreatic trypsin inhibitor (BPTI). Using a copper-beryllium high-pressure cell, we have performed small angle neutron scattering (SANS) experiment on NEAT spectrometer at HMI (Berlin, Germany). In the SANS configuration, the evolution of the radius of gyration and of the shape of the protein under pressures up to 6000 bar has been studied. When increasing pressure from atmospheric pressure up to 6000 bar, the pressure effects on the global structure of BPTI result on a reduction of the radius of gyration from 13.4 Å down to 12.0 Å. Between 5000 and 6000 bar, some transition already detected by FTIR [N. Takeda, K. Nakano, M. Kato, Y. Taniguchi, Biospectroscopy, 4, 1998, pp. 209–216] is observed. The pressure effect is not reversible because the initial value of the radius of gyration is not recovered after pressure release. By extending the range of wave-vectors to high q, we have observed a change of the form factor (shape) of the BPTI under pressure. At atmospheric pressure BPTI exhibits an ellipsoidal form factor that is characteristic of the native state. When the pressure is increased from atmospheric pressure up to 6000 bar, the protein keeps its ellipsoidal shape. The parameters of the ellipsoid vary and the transition detected between 5000 and 6000 bar in the form factor of BPTI is in agreement with the FTIR results. After pressure release, the form factor of BPTI is characteristic of an ellipsoid of revolution with a semi-axis a, slightly elongated with respect to that of the native one, indicating that the pressure-induced structural changes on the protein are not reversible. The global motions and the internal dynamics of BPTI protein have been investigated in the same pressure range by quasi-elastic neutron scattering experiments on IN5 time-of-flight spectrometer at ILL (Grenoble, France). The diffusion coefficients D and the internal relaxation times < τ₂> of BPTI deduced from the analysis of the intermediate scattering functions show a slowing down of protein dynamics when increasing pressure.

Keywords: BPTI; Protein Folding; Denatured States; Small Angle Neutron Scattering; Quasi-elastic Neutron Scattering; High Pressure

Incorporation of α-chymotrypsin into the 3D channels of bicontinuous cubic lipid mesophases by Julia Kraineva; Chiara Nicolini; Pappannan Thiyagarajan; Elena Kondrashkina; Roland Winter (pp. 424-433).

The effects of protein entrapment on the structure and phase behavior of periodically curved lipid mesostructures have been examined by synchrotron small-angle X-ray diffraction and FT-IR spectroscopy. The study was directed towards a better understanding of the effect of confinement in a lipid environment on the stability and unfolding behavior of α-chymotrypsin, and, vice versa, the effect of the entrapped protein on the lipid's mesophase structure and temperature- and pressure-dependent phase behavior. We compare the interaction of protein molecules of two different sizes (cytochrome c, 12.4 kDa, and α-chymotrypsin, 25.8 kDa) with the cubic Ia3d phase of monoolein (MO), which forms spontaneously in water. The cubic structure changes significantly when cyt c is incorporated: above a protein concentration of 0.2 wt.%, the interaction between the positively charged protein and the lipid headgroups leads to an increase in interfacial curvature which promotes the formation of a new micellar cubic phase, presumably of crystallographic space group P4₃32, which the lipid system does not form on its own. The larger α-chymotrypsin leads to a different scenario. On the basis of an examination of the calculated geometric parameters and water volume fractions, it is concluded that the α-chymotrypsin molecules cannot be located exclusively in the water channels of the cubic Ia3d or P4₃32 phases, but rather form new, less ordered (presumably cubic Pn3m) structures. The new structure disappears above the unfolding temperature of chymotrypsin and exhibits a pressure stability, which – in contrast to cyt c in MO – decreases with increasing chymotrypsin concentration in the system. While the secondary structure of cyt c remains unaffected in the confining lipid environment, the structure of α-chymotrypsin gets destabilized slightly, and the protein tends to aggregate even at relatively low concentrations.

Keywords: High pressure; SAXD; FT-IR; Lipid; Cubic phase; Monoolein; α-chymotrypsin; Cytochrome; c

Development of a low-pressure diamond anvil cell and analytical tools to monitor microbial activities in situ under controlled P and T by Phil M. Oger; Isabelle Daniel; Aude Picard (pp. 434-442).

We have designed a new low-pressure Diamond Anvil Cell (DAC), calibrated two novel pressure calibrants and validated the use of semi-quantitative Raman and X-ray spectroscopies to monitor the fate of microbes, their metabolism or their cellular components under controlled pressures and temperatures in the 0.1–1.4 GPa and 20–300 °C P, T range. The low-pressure DAC has a 250- to 600-μm-thick observation diamond window to allow for lower detection limits and improved microscopic imaging. This new design allows the determination of cellular growth parameters from automated image analysis, which can be correlated with the spectroscopic data obtained on metabolism, ensuring high quality data collection on microbial activity under pressure. The novel pressure sensors offer the ease of use of the well-known ruby scale, while being more sensitive and reacting to pressure variations instantaneously.

Keywords: Diamond Anvil cell; GFP; Yeast; High pressure; Cell cycle; Metabolism; DMR; Agrobacterium

Protein folding and aggregation: Two sides of the same coin in the condensation of proteins revealed by pressure studies by Jerson L. Silva; Yraima Cordeiro; Debora Foguel (pp. 443-451).

Hydrostatic pressure can be considered as “thermodynamic tweezers�? to approach the protein folding problem and to study the cases when folding goes wrong leading to the protein folding disorders. The main outcome of the use of high pressure in this field is the stabilization of folding intermediates such as partially folded conformations, thus allowing us to characterize their structural properties. Because partially folded intermediates are usually at the intersection between productive and off-pathway folding, they may give rise to misfolded proteins, aggregates and amyloids that are involved in many neurodegenerative diseases, such as transmissible spongiform encephalopathies, Alzheimer's disease, Parkinson's disease and Huntington's disease. Of particular interest is the use of hydrostatic pressure to unveil the structural transitions in prion conversion and to populate possible intermediates in the folding/unfolding pathway of the prion protein. The main hypothesis for prion diseases proposes that the cellular protein (PrP^C) can be altered into a misfolded, β-sheet-rich isoform, the PrP^Sc (from scrapie). It has been demonstrated that hydrostatic pressure affects the balance between the different prion species. The last findings on the application of high pressure on amyloidogenic proteins will be discussed here as regards to their energetic and volumetric properties. The use of high pressure promises to contribute to the identification of the underlying mechanisms of these neurodegenerative diseases and to develop new therapeutic approaches.

Keywords: Abbreviations; HHP; high hydrostatic pressure; PFD; protein folding disorders; IB; inclusion bodies; TTR; transthyretin; p53C; core domain of the tumor suppressor protein p53; α-syn; α-synucleinHydrostatic pressure; Prion; Neurodegenerative disease; Amyloidogenic protein; Protein folding; Parkinson's disease

Probing the pressure–temperature stability of amyloid fibrils provides new insights into their molecular properties by Filip Meersman; Christopher M. Dobson (pp. 452-460).

A number of medical disorders, including Alzheimer's disease and type II diabetes, is characterised by the deposition of amyloid fibrils in tissue. The insolubility and size of the fibrils has largely precluded the determination of their structures at high resolution. Studies probing the stability of amyloid fibrils can reveal which non-covalent interactions are important in the formation and maintenance of the fibril structure. In particular, we review here the use of high hydrostatic pressure and high temperature as perturbation techniques. In general, small aggregates formed early in the assembly process can be dissociated by high pressure, but mature amyloid fibrils are highly pressure stable. This finding suggests that a temporal transition occurs during which side chain packing and hydrogen bond formation are optimised, whereas the hydrophobic effect and electrostatic interactions play a dominant role in the early stages of the aggregation. High temperatures, however, can disrupt most aggregates. Though the observed stability of amyloid fibrils is not unique to these structures, the notion that amyloid fibrils can represent the global minimum in free energy is supported by this type of investigations. Some implications regarding the nature of toxic species, associated with at least many of the amyloid disorders, and recently proposed structural models are discussed.

Keywords: Abbreviations; HHP; high hydrostatic pressure; DMSO; dimethylsulphoxide; HT; high temperature; HFIP; 1,1,1,3,3,3-hexafluoro-2-propanol; TFE; 2,2,2-trifluoroethanol; SDS; sodium dodecyl sulphate; TTR; transthyretin; β; ₂; m; β; ₂; -microglobulin; AFM; atomic force microscopy; H/D; hydrogen-deuterium; FTIR; Fourier transform infrared; PrP; ^Sc; scrapie isoform of the prion protein; CH3CN; acetonitrile; HCOOH; formic acid; ANS; 8-anilino-naphthalene-1-sulphonatePressure; Dissociation; Precursor; Temperature; Structure

Pressure as a tool to study protein-unfolding/refolding processes: The case of ribonuclease A by M. Ribó; J. Font; A. Benito; J. Torrent; R. Lange; M. Vilanova (pp. 461-469).

This paper gives an overview of the application of high-pressure to study the folding/unfolding processes of proteins using Ribonuclease A as a model protein. A particular focus is the study of pressure-equilibrium unfolding and folding kinetics using variants and the information obtained by comparing these with the wild-type enzyme.

Keywords: High pressure; Ribonuclease A; Folding process; Site-directed mutagenesis; Protein engineering analysis

Tuning amyloidogenic conformations through cosolvents and hydrostatic pressure: When the soft matter becomes even softer by Wojciech Dzwolak (pp. 470-480).

Compact packing, burial of hydrophobic side-chains, and low free energy levels of folded conformations contribute to stability of native proteins. Essentially, the same factors are implicated in an even higher stability of mature amyloid fibrils. Although both native insulin and insulin amyloid are resistant to high pressure and influence of cosolvents, intermediate aggregation-prone conformations are susceptible to either condition. Consequently, insulin fibrillation may be tuned under hydrostatic pressure or – through cosolvents and cosolutes – by preferential exclusion or binding. Paradoxically, under high pressure, which generally disfavors aggregation of insulin, an alternative “low-volume�? aggregation pathway, which leads to unique circular amyloid is permitted. Likewise, cosolvents are capable of preventing, or altering amyloidogenesis of insulin. As a result of cosolvent-induced perturbation, distinct conformational variants of fibrils are formed. Such variants, when used as templates for seeding daughter generations, reproduce initial folding patterns regardless of environmental biases. By the close analogy, this suggests that the “prion strains�? phenomenon may mirror a generic, common feature in amyloids. The susceptibility of amyloidogenic conformations to pressure and cosolvents is likely to arise from their “frustration�?, as unfolding results in less-densely packed side-chains, void volumes, and exposure of hydrophobic groups. The effects of cosolvents and pressure are discussed in the context of studies on other amyloidogenic protein models, amyloid polymorphism, and “strains�?.

Keywords: Abbreviations; AFM; Atomic Force Microscopy; ASA; Accessible Surface Area; CD; Circular Dichroism; DAC; Diamond Anvil Cell; DSC; Differential Scanning Calorimetry; EtOH; Ethanol; FT-IR; Fourier Transform Infrared Spectroscopy; Ig-LC; Immunoglobulin Light Chain; NMR; Nuclear Magnetic Resonance; PPC; Pressure Perturbation Calorimetry; PrP; ^C; Native Cellular Isoform of Prion Protein; PrP; ^Sc; Scrapie Isoform of Prion Protein; TTR; Transthyretin; [URE3], [PSI; ⁺; ]; ‘yeast prions’—heritable traits in; Saccharomyces cerevisiae; formed by cellular proteins: Ure2p and Sup35p, respectivelyProtein aggregation; Insulin; Amyloid; High pressure; Cosolvent; Preferential hydration; Seeding

Folding studies of two hydrostatic pressure sensitive proteins by Cui-Yan Tan; Chun-He Xu; Kang-Cheng Ruan (pp. 481-488).

High hydrostatic pressure combined with various spectroscopies is a powerful technique to study protein folding. An ideal model system for protein folding studies should have the following characteristics. (1) The protein should be sensitive to pressure, so that the protein can be unfolded under mild pressure. (2) The folding process of the protein should be easily modulated by several chemical or physical factors. (3) The folding process should be easily monitored by some spectroscopic parameters. Here, we summarized the pressure induced folding studies of two proteins isolated from spinach photosystem II, namely the 23-kDa and the 33-kDa protein. They have all the characteristics mention above and might be an ideal model protein system for pressure studies.

Keywords: High pressure; Unfolding/folding of protein; Pressure jump; Protein stabilization

The use of pressure-jump relaxation kinetics to study protein folding landscapes by Joan Torrent; Josep Font; Heinz Herberhold; Stéphane Marchal; Marc Ribó; Kangcheng Ruan; Roland Winter; Maria Vilanova; Reinhard Lange (pp. 489-496).

Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refolding. These processes can be initiated by upward and downward pressure-jumps of amplitudes of a few 10 to 100 MPa, with a dead-time on the order of milliseconds. In many cases, the relaxation times can be easily determined when the pressure cell is connected to a spectroscopic detection device, such as a spectrofluorimeter. Adiabatic heating or cooling can be limited by small pressure-jump amplitudes and a special design of the sample cell. Here, we discuss the application of this method to four proteins: 33-kDa and 23-kDa proteins from photo-system II, a variant of the green fluorescent protein, and a fluorescent variant of ribonuclease A. The thermodynamically predicted equivalency of upward and downward pressure-jump induced protein relaxation kinetics for typical two-state folders was observed for the 33-kDa protein, only. In contrast, the three other proteins showed significantly different kinetics for pressure-jumps in opposite directions. These results cannot be explained by sequential reaction schemes. Instead, they are in line with a more complex free energy landscape involving multiple pathways.

Keywords: High pressure; Pressure jump; Protein folding; Relaxation kinetics

Refolding studies using pressure: The folding landscape of lysozyme in the pressure–temperature plane by L. Smeller; F. Meersman; K. Heremans (pp. 497-505).

Refolding of hen egg white lysozyme after pressure unfolding was measured by FTIR spectroscopy. The high-pressure treatment was found to be useful for unfolding/refolding studies because pressure acts against aggregation, and therefore no irreversible aggregation takes place during the pressure treatment. After the release of the pressure, folding intermediate structures were found which were formed during the decompression of the lysozyme. These were aggregation prone when heated, as indicated by their lower stability against aggregation. The intermediates were only formed if the protein was unfolded, subdenaturing pressures could not populate these intermediates. We introduced the notion of a superfunnel to describe the free energy landscape of interacting polypeptide chains. This can explain the propensity of folding intermediates to aggregate. A possible Gibbs-free energy landscape for lysozyme was constructed for the whole pressure–temperature plane.

Keywords: Folding; Lysozyme; Funnel; Free energy landscape; High pressure

Pressure denaturation of apomyoglobin: A molecular dynamics simulation study by Andrés N. McCarthy; J. Raúl Grigera (pp. 506-515).

The effect of pressure on the structure and mobility of Sperm Wale Apomyoglobin was studied by Molecular Dynamics computer simulation at 1 bar and 3 kbar (1 atm=1.01325 bar=101.325 kPa). The results are in good agreement with the available experimental data, allowing further analysis of other features of the effect of pressure on the protein solution. From the analysis of Secondary Structures (SS) along the trajectories it is observed that α-helixes are favoured under pressure at the expense of bends, turns and 3-helixes. The studies of mobility show that although the general mobility is restricted under pressure this is not true for some particular residues. The studies of tertiary structure show important conformational changes. The evolution of the Solvent Accessed Surface (SAS) with pressure shows a notorious increase due almost completely to a biased raise in the hydrophobic area exposed, which consequently shows that the hydrophobic interaction is considerably weaker under high hydrostatic pressure conditions.

Keywords: Pressure; Denaturation; Apomyoglobin

High pressure reveals that the stability of interdimeric contacts in the R- and T-state of HbA is influenced by allosteric effectors: Insights from computational simulations by I. Kövesi; G. Schay; T. Yonetani; M. Laberge; J. Fidy (pp. 516-521).

The molecular details of the mechanism of action of allosteric effectors on hemoglobin oxygen affinity are not clearly understood. The global allostery model proposed by Yonetani et al. suggests that the binding of allosteric effectors can take place both in the R and T states and that they influence oxygen affinity through inducing global tertiary changes in the subunits. Recently published high pressure studies yielded dissociation constants at atmospheric pressure that showed a stabilizing effect of heterotropic allosteric effectors on the dimer interface in the R state, and a more pronounced destabilizing effect in a T state model. In the present work, we report on computational modeling used to interpret the high pressure experimental data. We show structural changes in the hemoglobin interdimeric interfaces, indicative of a global tertiary structural change induced by the binding of allosteric effectors. We also show that the number of water molecules bound at the interface is significantly influenced by binding effectors in the T state in accordance with the experimental data. Our results suggest that the binding of effectors at definite sites leads to tertiary changes that propagate to the interfaces and results in overall structural re-organizations.

Keywords: Abbreviations; Hb; human hemoglobin A; IHP; inositol hexaphosphate; oxyHb; oxygenated Hb; α-oxyFe-β-ZnHb; (α Fe-O; ₂; ); ₂; -(β zinc-protoporphyrin-IX); ₂; DPG; 2,3-diphosphoglycerateHuman hemoglobin; Hb allostery; Heterotropic effector; MD simulation; Dimer interface; High pressure

A molecular dynamics simulation of SNase and its hydration shell at high temperature and high pressure by Nikolai Smolin; Roland Winter (pp. 522-534).

Temperature- and pressure-induced unfolding of staphylococcal nuclease (SNase) was studied by Royer, Winter et al. using a variety of experimental techniques (SAXS, FT-IR and fluorescence spectroscopy, DSC, PPC, densimetry). For a more detailed understanding of the underlying mechanistic processes of the different unfolding scenarios, we have carried out a series of molecular dynamics (MD) computer simulations on SNase. We investigated the initial changes of the structure of the protein upon application of pressure (up to 5 kbar) and discuss volumetric and structural differences between the native and pressure pre-denatured state. Additionally, we have obtained the compressibility of the protein and hydration water and compare these data with experimental results. As water plays a crucial role in determining the structure, dynamics and function of proteins, we undertook a detailed analysis of the structure of the interfacial water and the protein–solvent H-bond network as well. Moreover, we report here also MD results on the temperature-induced unfolding of SNase. The time evolution of the protein volume and solvent accessible surface area during thermal unfolding have been investigated, and we present a detailed discussion of the temperature-induced unfolding pathway of SNase in terms of secondary and tertiary structural changes.

Keywords: Protein–solvent interaction; Molecular dynamics simulation; High pressure; Compressibility; Protein unfolding

The yeast prion protein Ure2: Structure, function and folding by Hui-Yong Lian; Yi Jiang; Hong Zhang; Gary W. Jones; Sarah Perrett (pp. 535-545).

The Saccharomyces cerevisiae protein Ure2 functions as a regulator of nitrogen metabolism and as a glutathione-dependent peroxidase. Ure2 also has the characteristics of a prion, in that it can undergo a heritable conformational change to an aggregated state; the prion form of Ure2 loses the regulatory function, but the enzymatic function appears to be maintained. A number of factors are found to affect the prion properties of Ure2, including mutation and expression levels of molecular chaperones, and the effect of these factors on structure and stability are being investigated. The relationship between structure, function and folding for the yeast prion Ure2 are discussed.

Keywords: Abbreviations; BSA; bovine serum albumin; CDNB; 1-chloro-2,4-dinitrobenzene; GdmCl; guanidine hydrochloride; GPx; glutathione peroxidase; GSH; glutathione; GSSG; oxidized glutathione; GST; glutathione transferase; CHP; cumene hydroperoxide; t; -BH; tert; -butyl hydroperoxide; ThT; Thioflavin TPrion; Amyloid; Glutathione transferase; Peroxidase; Protein Folding; Chaperone

Modulation of prion protein structure by pressure and temperature by Joan Torrent; Maria Teresa Alvarez-Martinez; Jean-Pierre Liautard; Reinhard Lange (pp. 546-551).

High pressure and temperature have been used efficiently to shed light on prion protein structure and folding. These physical parameters induce different conformational states of the prion protein, suggesting that prion structural changes occur within a complex energy landscape. Pressure has been used to prevent and even reverse prion protein aggregation. Alternatively, depending on experimental conditions, pressure also promotes prion protein aggregation leading to the formation of amorphous aggregates and amyloid fibrils. The latter ones show all characteristics of the pathogenic scrapie form. Furthermore, the pressure effects on prion protein structure appear to be strongly dependent on the integrity of the disulfide bond. In this paper, we discuss the mechanism and the origin of these opposing effects of pressure, taking the truncated form of hamster prion protein (SHaPrP_90–231) as a model.

Keywords: Abbreviations; PrP; prion protein; PrP; ^C; cellular isoform; PrP; ^Sc; pathogenic isoform SHaPrP; _90–231; , Syrian hamster recombinant PrP consisting of residues 90–231; NMR; nuclear magnetic resonance; UV; ultraviolet; TEM; trasmission electron microscopy. ThT, thioflavinT; ANS; 8-anilino-1-naphthalene sulfonate; GdnHCl; guanidine hydrochloride; FTIR; Fourier transform infrared; DTT; dithiothreitolPrion protein; High pressure; Protein folding; Thermodynamic stability; Amyloid fibril

Protein conformation determines the sensibility to high pressure treatment of infectious scrapie prions by Philipp Heindl; Avelina Fernández García; Peter Butz; Eberhard Pfaff; Bernhard Tauscher (pp. 552-557).

Application of high pressure can be used for gentle pasteurizing of food, minimizing undesirable alterations such as vitamin losses and changes in taste and color. In addition, pressure has become a useful tool for investigating structural changes in proteins. Treatments of proteins with high pressure can reveal conformations that are not obtainable by other physical variables like temperature, since pressure favors structural transitions accompanied with smaller volumes. Here, we discuss both the potential use of high pressure to inactivate infectious TSE material and the application of this thermodynamic parameter for the investigation of prion folding. This review summarizes our findings on the effects of pressure on the structure of native infectious scrapie prions in hamster brain homogenates and on the structure of infectious prion rods isolated from diseased hamsters brains. Native prions were found to be pressure sensitive, whereas isolated prions revealed an extreme pressure-resistant structure. The discussion will be focused on the different pressure behavior of these prion isoforms, which points out differences in the protein structure that have not been taken into consideration before.

Keywords: Abbreviations; PrP; ^C; cellular isoform of the prion protein; PrP; ^Sc; pathogenic isoform of the prion protein; PrP27-30, N-truncated PrP; ^Sc; proteinase K-resistant and infectious core of PrP; ^Sc; iPrP27–30; isolated and resuspended PrP27–30; PK; Proteinase KPrion protein; High pressure; Inactivation; Proteinase K sensitivity; Prion conformation; Protein folding

Inactivation of transmissible spongiform encephalopathy agents in food products by ultra high pressure–temperature treatment by Franco Cardone; Paul Brown; Richard Meyer; Maurizio Pocchiari (pp. 558-562).

Bovine spongiform encephalopathy (BSE) contamination of the human food chain most likely resulted from nervous system tissue in mechanically recovered meat used in the manufacture of processed meats. The availability of effective decontamination methods for products considered at risk for BSE or other transmissible spongiform encephalopathies (TSEs) would be an attractive safeguard to human health, but neither of the two proven inactivating methods, autoclaving or exposure to strong alkali or bleach, are applicable to foodstuffs. Ultra high pressure–temperature treatment of foods is an effective decontamination method that can reduce the pathogen load while keeping unaltered the nutritional and organoleptic properties of the product. The application of different combinations of high pressure–temperature pulses to meat products ‘spiked’ with the agents of TSEs can reduce the level of infectivity by 10³ to 10⁶ mean lethal doses (LD₅₀) per gram of tissue. These data indicate that the high pressure–temperature treatment is a ready-to-use and feasible strategy to reduce the risk of TSEs transmission via contaminated meat products.

Keywords: Abbreviations; BSE; bovine spongiform encephalopathy; TSE; transmissible spongiform encephalopathy; vCJD; variant Creutzfeldt–Jakob disease; BASE; bovine amyloidotic spongiform encephalopathy; LD; ₅₀; mean lethal dose; PrPres; protease-resistant pathological prion proteinTransmissible spongiform encephalopathy; Prion disease; Prion protein; Inactivation method; Food safety; High pressure–temperature treatment

Pressure and temperature as tools for investigating the role of individual non-covalent interactions in enzymatic reactions by Emanuela Occhipinti; Nicole Bec; Benedetta Gambirasio; Gabriella Baietta; Pier Luigi Martelli; Rita Casadio; Claude Balny; Reinhard Lange; Paolo Tortora (pp. 563-572).

Sulfolobus solfataricus carboxypeptidase, (CPSso), is a heat- and pressure-resistant zinc-metalloprotease. Thanks to its properties, it is an ideal tool for investigating the role of non-covalent interactions in substrate binding. It has a broad substrate specificity as it can cleave any N-blocked amino acid (except for N-blocked proline). Its catalytic and kinetic mechanisms are well understood, and the hydrolytic reaction is easily detectable spectrophotometrically. Here, we report investigations on the pressure- and temperature-dependence of the kinetic parameters (turnover number and Michaelis constant) of CPSso using several benzoyl- and 3-(2-furyl)acryloyl-amino acids as substrates. This approach enabled us to study these parameters in terms of individual rate constants and establish that the release of the free amino acid is the rate-limiting step, making it possible to dissect the individual non-covalent interactions participating in substrate binding. In keeping with molecular docking experiments performed on the 3D model of CPSso available to date, our results show that both hydrophobic and energetic interactions (i.e., stacking and van der Waals) are mainly involved, but their contribution varies strongly, probably due to changes in the conformational state of the enzyme.

Keywords: Sulfolobus solfataricus; Carboxypeptidase; High pressure; Enzyme catalysis; Non-covalent interaction

Hydrostatic and osmotic pressure study of the hairpin ribozyme by Guy Hervé; Sylvia Tobé; Thomas Heams; Jacques Vergne; Marie-Christine Maurel (pp. 573-577).

The recent discovery of numerous catalytically active RNAs in various living species as well as the in vitro selection of a large series of RNA aptamers able to bind specifically various molecules such as metabolites and co-factors, emphasize the adaptability of RNAs through the plasticity of their secondary structure. Furthermore, all these observations give support to the “RNA world�? hypothesis as a step in the primitive development of life on Earth. On this background, we used high pressure to study the mechanism of action of a model hairpin ribozyme which exhibits self-cleavage and ligation. The activation volume (Δ V^≠) of the cleavage reaction (34±4 ml/mol) indicates that an important compaction of the RNA molecule occurs during the reaction and must be accompanied by a significant movement of water molecules . Indeed, such a release of 78±4 water molecules per RNA molecule could be measured by complementary osmotic shock experiments. These results are consistent with the information provided by the structural studies which indicate that two loops of the RNA molecule should come into contact for the reaction to occur .The high pressure study of a modified form of the ribozyme whose activity is strictly dependent on the presence of adenine as a co-factor should bring some information about the structural significance of this important Δ V^≠ of activation.

Keywords: High pressure; Osmotic pressure; Hairpin ribozyme; RNA world

High-pressure studies of the reaction mechanism of nitric-oxide synthase by Antonius C.F. Gorren; Stéphane Marchal; Morten Sørlie; K. Kristoffer Andersson; Reinhard Lange; Bernd Mayer (pp. 578-585).

Nitric-oxide synthase (NOS) generates nitric oxide froml-arginine in two reaction cycles with N^ω-hydroxy-l-arginine as an obligate intermediate. Although much progress has been made in recent years in the elucidation of the reaction mechanism of NOS, many questions remain to be answered. The use of low temperature has been instrumental in the revelation of the mechanism of NO synthesis, particularly regarding the role of the cofactor 5,6,7,8-tetrahydrobopterin (BH4). High-pressure studies may be expected to be similarly useful, but have been very few so far. In this short review, we depict the present state of knowledge about the reaction mechanism of NO synthesis, and the role(s) BH4 plays in it. This exposition is followed by a summary of the results obtained thus far in high-pressure studies and of the conclusions that can be drawn from them.

Keywords: Abbreviations; NOS; nitric oxide synthase; eNOS, nNOS, and iNOS; endothelial, neuronal, and inducible isoforms of NOS; eNOS; _oxy; isolated oxygenase domain of (bovine) eNOS; NHA; N; ^ω; -hydroxy-; l; -arginine; BH4; tetrahydrobiopterin ((; 6R; )-5,6,7,8-tetrahydro-6-(; l; -; erythro; -1′,2′-dihydroxypropyl)pterin); ^BH3; 6,7,8-trihydrobiopterin radical; BH2; 7,8-dihydrobiopterin; 4-Am-BH4; 4-amino-tetrahydrobiopterin ((; 6R; )-2,4-diamino-5,6,7,8-tetrahydro-6-(; l; -; erythro; -1′,2′-dihydroxypropyl)pteridine); ^4-Am-BH3; and 4-Am-BH2; ^{see BH3}; and BH2; EPR; electron paramagnetic resonance; UV/Vis; ultraviolet visibleNitric-oxide synthase; High pressure; Tetrahydrobiopterin; Heme ligand binding

The pressure effect on the structure and functions of protein disulfide isomerase by Kazuyoshi Ado; Naohiro Takeda; Masakazu Kikuchi; Yoshihiro Taniguchi (pp. 586-592).

Studying on the pressure effects of the structure and functions of the multidomain protein, protein disulfide isomerase (PDI), the intrinsic Trp fluorescence spectra of PDI were measured under high pressure. PDI has 5 Trp residues and the two of all Trp residues are located at the neighborhood of the active site (WCGHC) for isomerase activity. On the basis of the red shift of center of spectral mass (CSM) of the intrinsic Trp fluorescence and the decrease in its fluorescence intensity, the changes in tertiary structure of PDI were observed above 100 MPa. These structural changes were completed at 400 MPa. The CSM of 400 MPa denatured PDI was comparable to that of 6.0 M GdnHCl denatured one. All of the Trp residues included in PDI are completely exposed to aqueous medium at 400 MPa. However, there is the significant difference between the pressure and GdnHCl-denatured PDI. The Trp fluorescence intensity was decreased with increasing pressure, but increased with the increase of the GdnHCl concentration. It is implied that the pressure-denatured state of PDI might remain compact not to be extensively unfolded. In the point of view about the reversibility of pressure-treated PDI, the tertiary structure was completely recovered after released to ambient pressure. The disulfide reduction and chaperone activity of 400 MPa-treated PDI were also recovered to be comparable to those of native one. Despite of a multidomain protein, the excellence in both structural and functional recovery of pressure-denatured PDI is quite remarkable. These unique properties of PDI against high pressure provide the insights into understanding the pressure-induced denaturation of PDI.

Keywords: Abbreviations; PDI; protein disulfide isomerase; Trp; tryptophan; CSM; the center of spectral mass; GdnHCl; guanidine hydrochloride; SDS-PAGE; sodium dodecyl sulfate poly acrylamide gel electrophoresis; GSH; glutathione reduced form; GSSG; glutathione oxidized form; GR; glutathione reductase; NADPH; β-nicotinamide adenine dinucleotide phosphate, reducedProtein disulfide isomerase; High pressure; Tryptophan fluorescence; Volume change; Reduction activity; Chaperone activity

High pressure-induced changes in bovine milk proteins: A review by Thom Huppertz; Patrick F. Fox; Kees G. de Kruif; Alan L. Kelly (pp. 593-598).

High pressure (HP)-induced changes in the proteins of bovine milk have become an area of considerable research interest in recent years; as a result, there is now a detailed understanding of the effects of HP on casein micelles and whey proteins. HP treatment at pressures >400 or >100 MPa denatures the two most abundant whey proteins, α-lactalbumin (α-la) and β-lactoglobulin (β-lg), respectively. The majority of denatured β-lg in HP-treated milk associates with the casein micelles, although some denatured β-lg remains in the serum phase or is attached to the milk fat globule membrane; HP-denatured α-la is also associated with the milk fat globules. Casein micelles are disrupted on treatment at pressures >200 MPa; the rate and extent of micellar disruption increases with pressure and is probably due to the increased solubility of calcium phosphate with increasing pressure. On prolonged treatment at 250–300 MPa, reassociation of micellar fragments occurs through hydrophobic bonding; this process does not occur at a pressure >300 MPa, leading to considerably smaller micelles in such milk. As a result of HP-induced changes, the size, number, hydration, composition and light-scattering properties of casein micelles in HP-treated milk differ considerably from those in untreated milk.

Keywords: High pressure; Milk; Casein micelle; α-lactalbumin; β-lactoglobulin

High pressure–low temperature processing of food proteins by Eliane Dumay; Laetitia Picart; Stéphanie Regnault; Maryse Thiebaud (pp. 599-618).

High pressure–low temperature (HP–LT) processing is of interest in the food field in view of: (i) obtaining a “cold�? pasteurisation effect, the level of microbial inactivation being higher after pressurisation at low or sub-zero than at ambient temperature; (ii) limiting the negative impact of atmospheric pressure freezing on food structures. The specific effects of freezing by fast pressure release on the formation of ice I crystals have been investigated on oil in water emulsions stabilized by proteins, and protein gels, showing the formation of a high number of small ice nuclei compared to the long needle-shaped crystals obtained by conventional freezing at 0.1 MPa. It was therefore of interest to study the effects of HP–LT processing on unfolding or dissociation/aggregation phenomena in food proteins, in view of minimizing or controlling structural changes and aggregation reactions, and/or of improving protein functional properties. In the present studies, the effects of HP–LT have been investigated on protein models such as (i) β-lactoglobulin, i.e., a whey protein with a well known 3-D structure, and (ii) casein micelles, i.e., the main milk protein components, the supramolecular structure of which is not fully elucidated. The effects of HP–LT processing was studied up to 300 MPa at low or sub-zero temperatures and after pressure release, or up to 200 MPa by UV spectroscopy under pressure, allowing to follow reversible structural changes. Pressurisation of ∼2% β-lactoglobulin solutions up to 300 MPa at low/subzero temperatures minimizes aggregation reactions, as measured after pressure release. In parallel, such low temperature treatments enhanced the size reduction of casein micelles.

Keywords: High pressure; Low or sub-zero temperature; Water phase diagram; Milk protein; β-lactoglobulin; Casein micelle

High pressure application for food biopolymers by Dietrich Knorr; Volker Heinz; Roman Buckow (pp. 619-631).

High hydrostatic pressure constitutes an efficient physical tool to modify food biopolymers, such as proteins or starches. This review presents data on the effects of high hydrostatic pressure in combination with temperature on protein stability, enzymatic activity and starch gelatinization. Attention is given to the protein thermodynamics in response to combined pressure and temperature treatments specifically on the pressure–temperature–isokineticity phase diagrams of selected enzymes, prions and starches relevant in food processing and biotechnology.

Keywords: High pressure; Phase diagrams; Enzyme; Starch; Prion

What lies in the future of high-pressure bioscience? by Claude Balny (pp. 632-639).

Without being comprehensive in this mini-review, I will address perspectives, some speculative, for the development and use of high pressure to explore biochemical phenomena. This will be illustrated with several examples.

Keywords: Abbreviations; NMR; nuclear magnetic resonance; AFM; atomic force microscopy; HAV; hepatitis A virusHigh pressure; Biotechnology; Folding; Enzymology; Protein compressibility; Energy landscape; Prion; Amyloid

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BBA - Proteins and Proteomics (v.1764, #3)