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High hydrostatic pressure: a probing tool and a necessary parameter in biophysical chemistry Filip Meersmanabc and Paul F. McMillan*d High pressures extending up to several thousands of atmospheres provide extreme conditions for biological organisms to survive. Recent studies are investigating the survival mechanisms and biological function of microorganisms under natural and laboratory conditions extending into the GigaPascal

Received 31st July 2013, Accepted 24th October 2013

range, with applications to understanding the Earth’s deep biosphere and food technology. High

DOI: 10.1039/c3cc45844j

and functional properties of biologically important macromolecules and polymers encountered in soft

pressure has also emerged as a useful tool and physical parameter for probing changes in the structure matter science. Here we highlight some areas of current interest in high pressure biophysics and

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physical chemistry that are emerging at the frontier of this cross-disciplinary field.

Introduction High pressure investigations are typically associated with Earth and planetary sciences or materials research under extreme conditions, as solids and liquids are studied up to pressures into the multimegabar range (>100 GPa) (Fig. 1). However, equally significant work is also being carried out on important macromolecules and biological organisms in a range extending up to a few thousand atmospheres (100–1000 MPa). These constitute a corresponding set of extreme conditions for such ‘‘soft’’ matter systems. Although studies of this type date back to the earliest days of high pressure research, they now constitute a rapidly expanding field of interdisciplinary research with applications extending from understanding the Earth’s deep biosphere and possibly origins of life, to food technology, nanoscale materials science and medicine. Here we describe some recent advances and areas in which high pressure biophysical chemistry studies are playing a key role in understanding structural transformations in macromolecules and their biologically relevant properties. A key concept underpinning all pressure-related phenoˆtelier’s principle that states that any reacmena is Le Cha tion or physical transformation that is accompanied by a reduction in volume will be favoured at high pressure.1 a

Department of Chemistry, University College London, 20 Gordon St., London WC1H 0AJ, UK. E-mail: [email protected] b Rousselot-Expertise Centre, R&D Laboratory, Meulestedekaai 81, 9000 Gent, Belgium. E-mail: [email protected]; Fax: +32 (0)9255 6551; Tel: +32 (0)9255 1826 c Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium d Department of Chemistry, University College London, 20 Gordon St., London WC1H 0AJ, UK. E-mail: [email protected]; Tel: +44 (0)20 7679 4610

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Thermodynamically this is expressed by:   @ ln K DV ¼ @P T RT

(1)

where K is the equilibrium constant, P the pressure, R the universal gas constant, T the absolute temperature and DV the volume change, which is the pressure derivative of the change in free energy (DG) at constant temperature. The pressure dependence of the volume change is given by the isothermal compressibility bT. In inorganic systems this expression governs reactions and phase transformations among solids, liquids and gases. Biochemical phenomena including the disruption of biomolecular interactions, unfolding of proteins and lipid phase transitions also obey this rule. The conditions for a negative volume change to occur include the presence of void volumes and differential hydration states.2–4 In many cases, the origin of the volume change is still unclear and this provides an active area of research motivating high pressure studies. Pressure also has dramatic effects on the kinetics of reactions and structural transformations, as the volumes of transition or intermediate states can be greater or less than those of the initial conformations or reactants. Kinetic studies carried out under high pressure conditions provide an important tool to understand transition pathways and reaction mechanisms.5–7 A useful observation that is sometimes elevated to the status of a general principle is that of microscopic ordering, implying that at a given temperature a pressure increase results in a lowered entropy of the system.8,9 In this way pressure is considered to have an inverse effect to temperature and this general relationship has been observed to apply in biomacromolecular systems such as myoglobin.10 In inorganic and nonbiological systems application of pressure is often observed to aid in crystallisation processes and development of crystalline

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order and the pressure variable has been exploited to obtain better diffraction patterns in protein crystallography.11 However, the pressure-ordering principle does not always hold true leading to interesting phenomena in physical chemistry as well as biomolecular science. For example, crystals often exhibit ranges over which the melting relation (dTm/dP) exhibits a negative slope so that the crystal–liquid transformation occurs during pressurisation at constant temperature. The related phenomenon of pressure-induced amorphisation is observed to occur during compression at low temperature for a wide range of substances.12–14 Both of these phenomena can be mapped on to analogous transformations among soft matter and biomacromolecular systems, providing important information on the topology and transformation pathways of the configurational energy landscape.14–17 Pioneering experiments both in polymer science and high pressure biochemistry were carried out by Percy W. Bridgman, generally recognised as the father of modern high pressure research.18–21 In 1914 he published a landmark paper on ‘‘The coagulation of albumen by pressure’’.22 In his typically understated first sentence he writes: ‘‘The purpose of this note is to state a fact of possible biological interest which I have discovered incidentally in the course of other work’’. He then proceeds to show that pressure induces coagulation of egg white, effectively implying the existence of pressure-induced protein unfolding, that it is easier to induce this effect at lower temperatures, and that the pressure-induced coagulation event

may differ from that obtained by heating egg white. This classic one-page contribution thus describes some of the key features of pressure effects on proteins that form the basis for investigations to the present day, including key differences between the thermodynamic variables of heat and pressure in their effects on biomolecules. The main macromolecular components of cells include proteins, lipids and nucleic acids as well as sugars and polysaccharides. These compounds have all been studied extensively at high pressure and changes recorded in their structure and function as a function of the density have been reviewed elsewhere.1,23–28 Here we discuss examples of how pressure can be harnessed as a tool to address current issues in biophysics and biochemistry, including protein folding and dynamics, and mechanical properties of biomacromolecular assemblies. We also examine the role of pressure as a physical variable experienced by biological organisms, with recent studies contributing to our understanding of the deep biosphere and the emergence of life on Earth.29–31

Filip Meersman received his PhD in Biochemistry from the Katholieke Universiteit Leuven in 2002 for work on the effects of pressure on proteins. He then went on to join the group of Professor Christopher Dobson at the University of Cambridge as a Marie Curie Intra-European Fellow, where he studied the stability, structure and formation mechanism of amyloid fibrils. From 2005 to 2011 he was a Filip Meersman fellow of the Research Fund Flanders at the Katholieke Universiteit Leuven, where he pursued his research interests in the pressure–temperature phase behaviour of proteins, protein assemblies and water-soluble polymers. Since 2007 he is a honorary research associate at University College London affiliated with the group of Paul McMillan. He is also a research leader at the University of Antwerp.

Paul F. McMillan is Sir William Ramsay Professor of Chemistry at University College London. He graduated with a BSc in Chemistry from Edinburgh University in 1977 and obtained his PhD degree at Arizona State University (1981). He remained at ASU becoming full Professor in 1992, and Director of the Centre for Solid State Science in 1997. In 2000 he moved to London to occupy a new Chair in Solid Paul F. McMillan State Chemistry at UCL and the Royal Institution, before becoming Ramsay chair at UCL in 2008. In London, he established laboratories for solid state chemistry and materials research under high pressure-high temperature conditions, along with work on nanomaterials and amorphous materials including the unusual phenomena of density-driven liquid–liquid phase transitions and polyamorphism. Now his high pressure interests are extending into biophysics and biology. He received a Wolfson-Royal Society Research Merit Award (2001– 2006) and an EPSRC Senior Research Fellowship (2006–2011). He was elected Fellow of the Royal Society of Chemistry and received the RSC award for Solid State Chemistry in 2003, and the Peter Day award in 2011.

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Protein dynamics and excited states Recent studies have harnessed the power of high pressure research to investigate conformational states of proteins especially associated with the dynamical nature of their structure. The structural representations of proteins determined by X-ray

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Fig. 1 Pressure ranges encountered throughout the universe, highlighting those most relevant to biology and macromolecular science. The logarithmic vertical scale is shown in atmospheres (1 atm–1 bar): 1000 atm = 1 kbar = 0.1 GPa = 100 MPa. Two main types of structural transformation encountered in macromolecular science that determine biological processes are illustrated at right: pressure-induced denaturation of proteins (above) and the fluid–gel transition of lipid bilayers that constitute cell membranes. Note that these illustrations are not intended to be positioned relative to the main pressure axis shown at left. Typical pressure ranges for key events affecting the biosphere are listed at right.

crystallography tend to promote the view that these are static entities. However, that interpretation is challenged by experimental observations including the facts that proteins can undergo hydrogen–deuterium (H/D) exchange, that crystal structures of some proteins are found to be different with or without bound ligands, and that the fluorescence of excited fluorophores can be quenched by direct collision with a solute molecule (e.g. tryptophan quenching by O2).32,33 This last observation implies that oxygen must access the tryptophan in the protein interior, despite the lack of any apparent ‘channel’ connecting the medium surrounding the protein with the internal fluorophore. It is now well established that active backbone dynamics are essential for protein functionalities such as enzymatic activity and for molecular motors that convert chemical energy into mechanical work. Side chain dynamics also play a key role in ligand docking, for example. An important consequence from the viewpoint of energy landscape models is that the native state is not well represented by a single conformation, but rather by a global minimum containing an ensemble of nearly iso-energetic conformations, that are

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termed conformational substates or excited states. Although more unfolded conformations are rarely explored, some of these structures can be significantly different to the static ground state as found, for example, in the case of folding intermediates.34 Despite the importance of such protein excited states to our understanding of essential cellular events, they remain poorly characterised despite recent advances combining NMR, small angle X-ray scattering (SAXS) and computational approaches to study their structures.33–35 This is mainly due to their short lifetimes and the fact that at any time they only make up a very small fraction of the total native population.33 High pressure techniques using high resolution X-ray crystallography and NMR structure determination combined with other biophysical techniques are now being applied to provide a useful and simpler alternative approach to study the conformational substates.36–40 If these possess a smaller volume than the native ground state then applying pressure will increase their abunˆtelier’s dance within the overall population according to Le Cha principle (eqn (1)), making them easier to observe.41 Pioneering work carried out by Frauenfelder and co-workers used myoglobin as a model system to explore how oxygen and carbon monoxide can reach and bind to the iron ion of the heme group in absence of any ‘channel’ connecting the heme with the protein surroundings.42,43 Frauenfelder et al.42 followed the CO stretching vibration in the IR spectrum to demonstrate that lowered pH and increased pressure both induce a similar shift in substate populations from A0 to A1. Following that result Urayama et al.38 wondered whether such a population shift might be reflected in more global conformational changes that would be observable by X-ray crystallography. This group developed a technique in which myoglobin crystals were pressurised to 150 MPa and subsequently cooled in order to ‘‘freeze in’’ any conformational changes that had occurred. The crystals were then studied at ambient pressure and cryogenic temperatures. Comparison of the structures determined at pH 4 and 6 with those obtained at 0.1 MPa and 150 MPa confirmed that the effect of pressure was indeed similar to that of lowering the pH, indicating clearly that myoglobin substates are characterised by distinct global conformations that can be both promoted or induced by pressure changes and recovered by quenching at low temperature. Advances in high pressure crystallography have opened up new possibilities to explore biomolecular structures at high pressure.44 In a series of studies on urate oxidase (UOX), a homotetramer that catalyses the hydroxylation of uric acid in the presence of molecular oxygen, Colloc’h and co-workers showed that compressing the UOX crystals under oxygen pressures up to 4.0 MPa causes O2 to occupy a previously unidentified polar site.45 The active site in UOX can sequentially bind O2 and H2O at the same position, with implications for understanding the reaction mechanism. Further insight was gained in a subsequent investigation of the tetrameric crystal structure at 150 MPa.36 The observed structural changes at this pressure were related to the existence of a conformational substate in which the active site is enlarged at the expense of a

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neighbouring hydrophobic cavity. Oxygen-binding sites in proteins can also be identified indirectly using pressurised xenon (Xe) that has a similar size to O2.45 Xe incorporation within hydrophobic sites associated with folded proteins used as models for ion channel complex components has been used to help understand anaesthetic effects of rare gases and other chemically inert molecules.46–48 Electron paramagnetic resonance (EPR) spectroscopy has been used to study the effect of pressure on T4 lysozyme.37 Using site-directed spin labelling introducing a nitroxide side chain (R1) the presence of different conformations can be observed as different spectral components in the EPR spectrum. This method enables us to distinguish between conformational and rotameric substates, as it was found that the pressure-dependence of the equilibrium constant is non-linear in the former and linear in the latter case. The shift in rotamer population with pressure and the change in equilibrium constant are illustrated in Fig. 2. This figure shows two possible rotamer positions of R1 at position 44, where an interaction with the neighbouring glutamate at position 45 results in an immobilised R1 side chain, denoted as population i. Rotation over dihedral angle w4 yields a mobile side chain, denoted as population m. The EPR spectra show that as pressure increases the population of the immobilised state increases, indicating that this rotamer adopts a lower volume. The inset shows the linear dependence of ln (KP/K0) on pressure (with KP and K0 the equilibrium constants at pressure P and 0.1 MPa, respectively), indicating that in this case the difference in partial molar isothermal compressibility between the two rotamers is zero. It was also pointed out that EPR may provide a straightforward method to determine small differences in partial molar isothermal compressibility and that, because of the site-directed labelling, this information can be obtained in a sequence specific manner. The conformational dynamics of N-Ras, a membraneassociated protein involved in signaling, have been assessed by IR spectroscopy as a function of pressure in its different

Fig. 2 Effect of pressure on the EPR spectra of T4 lysozyme containing the R1 spin label in position 44. (A) Model of the R1 side chain showing the two rotamers m and i, as well as the neighbouring glutamate E45. (B) and (C) show the pressure dependence of the EPR spectra and the equilibrium constant, respectively. The EPR spectra vary from 0 to 4 kbar (1 kbar = 100 MPa) with the highest pressure point shown in red (smallest amplitude curve) (After McCoy & Hubbell.37).

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Fig. 3 Pressure dependence of the dynamic incoherent structure factor S(Q,o) of BPTI at 0.1 and 600 MPa (after Appavou et al.50).

nucleotide binding states in the presence or absence of a model membrane.49 Here it was found that both nucleotide binding and the presence of the membrane have a drastic effect on the conformational dynamics of N-Ras, and as a consequence also on the selection of conformational substates. In addition, a previously unobserved substate that appears upon membrane binding was found in this study. So far we have discussed the pressure-induced shift in native-like populations to access excited states or substates, making them amenable to structural characterisation. New information on protein dynamics can also be obtained from neutron and X-ray scattering experiments carried out at high pressure.50,51 Such studies indicate a general slowdown in dynamic processes under pressure, in agreement with MD simulations.52 Small angle X-ray scattering (SAXS) investigations of hen egg white lysozyme at pressures below the unfolding pressure show that an increase in density causes changes in the hydration layer surrounding the enzyme at around 150 MPa. Elastic and quasi-elastic neutron scattering (ENS and QENS) measurements then indicated that the dynamics of the protein slow down at a similar pressure, suggesting that the events are correlated.51 Similar results were obtained from QENS studies on bovine pancreatic trypsin inhibitor (BPTI).50 Fig. 3 shows the effect of increasing pressure on the dynamic incoherent structure factor S(Q,o) of BPTI. The observed signal contains an elastically scattered peak centred around zero energy transfer with quasi-elastic Lorentzian contributions reflecting the dynamics. The half-width at half-height of this signal decreases at high pressure, indicating a slowing down of the global and internal motions. Although the change in peak width is small, the difference is significant and corresponds to a major decrease in diffusion coefficient. The global diffusion coefficient was reported to decrease from 19  107 cm2 s1 at 0.1 MPa to 12  107 cm2 s1 at 600 MPa. This damping of protein motions at high pressure is reflected in a free energy landscape for proteins that is rougher than that at atmospheric pressure52 and it explains why folding is slower at elevated pressures compared to atmospheric pressure.1

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Kinetic experiments at high pressure Kinetic studies at high pressure provide fundamental insights into time-dependent phenomena such as protein folding, ligand binding and enzyme reactions.53 Classic stopped-flow measurements as well as pressure-jumps, that follow the relaxation processes following rapid pressure changes ranging up to 100 MPa in magnitude, can readily be performed at high pressure. If the sample volume is relatively large then one must typically take into account adiabatic heating or cooling effects for upward and downward P-jumps, respectively, resulting in temperature variations on the order of 0.1 1C per 10 MPa.53 Recently, Brooks and co-workers have developed a SAXS cell that allows both static and dynamic P-experiments in a wide T-range from 20 to +120 1C with P-jumps varying between 0.1 and 500 MPa.54 The small cell volume (3–11 mm3) makes adiabatic heating of the sample upon P-variation negligible. Moreover, the pressure change equilibrates immediately throughout the sample, without development of gradients.28 Important applications of P-jump and high pressure stopped-flow experiments have been made in the field of protein folding, where the determination of activation volumes for folding (DV #f) and unfolding (DV #u) processes provides information on the nature and hydration of the transition state ensemble (TSE). In the case of the ankyrin domain of the Notch receptor (Nank1–7) the folding and unfolding activation volumes are +50 and 18 mL mol1, respectively, indicating that the TSE is closer to the native than to the unfolded state on the reaction coordinate and that a large degree of dehydration occurs when the polypeptide chain adopts the TSE conformation.55 Similar results have been reported for several other proteins,6,56–58 whereas ribonuclease A (RNase A) was found to have a relatively wet TSE5 (Fig. 4). Jacob et al. performed both high pressure stopped flow and P-jump experiments and demonstrated that the activation volumes obtained by both techniques are in good agreement with each other.56 The kinetic profiles of folding–unfolding also give insight into the folding transition in terms of the possible involvement of intermediate conformations and the existence of slow reactions such as cis–trans isomerisation. This is illustrated for RNase A in Fig. 4 where the downward P-jump shows monophasic behaviour, whereas the upward P-jump is best described by biphasic kinetics, with the slow phase due to cis–trans isomerisation.5 At higher pressures the biphasic kinetics become monophasic. It has been noted that P-jump experiments provide an essential complement to temperature experiments as T-jumps cannot be performed in opposing directions.5 Likewise, one can also monitor the kinetics of aggregate dissociation and obtain useful information on the (de)polymerization mechanism and structural features of the aggregate.59,60 P-jump experiments also provide new insights into physiologically relevant reactions. Pressure-jumps as low as 20 MPa on microliter solutions have revealed details of the interaction of myosin subfragment-1 with adenosine diphosphate (ADP) that causes a conformational change affecting the myosin–actin interaction.61 This reaction underpins the force generating mechanism of myosin type molecular motors.

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Fig. 4 P-jump-induced folding/unfolding of Y115W RNase A at 30 1C and pH 5. Top: schematic representation of the energetics of the folding–unfolding reaction of RNase A: F is the native (folded) state, U the unfolded state and TSE the transition state ensemble. Bottom: relaxation kinetics of downward (A) and upward (B) P-jumps. The top and bottom X-axis values represent the time (s) profile of the downward and upward jumps, respectively. (C) is the upward P-jump fitted with a single exponential (From Font et al.5).

Synchrotron X-ray scattering and small angle neutron scattering (SANS) in combination with P-jump experiments offer a unique view of the time course of lipid phase transitions. For example, inverse bicontinuous cubic phases can occur in cellular processes involving the membrane, including endo/ exocytosis and membrane budding, and have potential applications in drug delivery.28,62 Squires et al.63 observed a gyroid (QGII) to diamond (QDII) bicontinuous cubic mesophase phase transition at 59.5 1C following a P-jump from 60 MPa to 24 MPa. Interestingly, they could also see a transient inverse hexagonal HII phase which is not observed in static experiments. This transient phase may in fact serve to accommodate the excess water during the transition, rather than being a true structural intermediate.23 In contrast, P-jumps of 50 to 150 MPa on the system monoolein : water (70 : 30 wt%) showed that the transition between the gyroid and diamond phases takes place without the formation of a detectable intermediate.62 In addition to providing structural and mechanistic insights into lipid phase transitions including activation parameters, such experiments can be used to provide important experimental tests of theoretically derived concepts and models.

Mechanical properties of biological assemblies Many biological polymers play a key role as structural materials. For instance, the exoskeleton of crustaceans is made of the

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polysaccharide chitin, whereas the cell walls of plants consist of cellulose. Proteins such as collagen are an important constituent of the extracellular matrix in skin and tendons. A feature shared by many of these polymers is their hierarchical character. There is growing interest in the mechanical properties of these polymers from a materials point of view.64,65 Understanding the mechanisms underlying their high mechanical strength and yield mechanisms not only gives insight into natural building materials; it also provides inspiration for the design of synthetic materials as well as composites containing biological polymers. Mechanical properties such as Young’s modulus are typically obtained from computer simulations or atomic force microscopy (AFM) experiments conducted under tensile stress conditions.64 Recently, using approaches adapted from high pressure materials research, the bulk modulus of cellulose and amyloid fibrils has been determined directly during compression experiments.66–68 The latter are hierarchical structures that originate from the self-assembly of proteins and that have been observed to possess interesting mechanical properties.64,69 Three principal reflections were recorded for amyloid fibrils formed from the transthyretin derived peptide TTR105115 by synchrotron X-ray diffraction. These provided the dimensions of a rectangular parallelepiped that was assumed to constitute the fundamental volume element and compression experiments in the diamond anvil cell allowed determination of V/V0 versus pressure. Equation of state (EOS) analysis then yielded bulk modulus values of 3–8 GPa. Determination of elastic modulus values in such compression experiments is free from the assumptions that must be applied to extract similar data from atomic force microscopy (AFM) investigations.69,70 The validity of the approach to analyse the fibril results was confirmed by applying the same methodology to obtain the bulk modulus of cellulose, for which the crystalline structure is known (Fig. 5).68 In that case it was demonstrated that the widely different values reported from X-ray diffraction vs. Raman spectroscopy experiments was due to the fact that each technique was probing different structural features. In the case of Raman scattering, the main peak followed during compression experiments was due to in-plane C–C stretching vibrations, whereas X-ray diffraction studied the ‘‘easier’’ compression between the planes.68 The amyloid fibril compression experiments were carried out using two different pressure-transmitting media, and this revealed valuable new structural information. First, by comparing the results obtained in water and in silicone oil it was demonstrated that the sheet–sheet interface is essentially dry. To our knowledge this is the first experimental confirmation of successful application of the dry zipper model proposed by Eisenberg and co-workers established using crystals of amyloidogenic peptides as models for details of the fibril structure.71 Secondly, the effect of pressure along the fibril axis was found to be quite different in silicone oil and in water, with the compressibility being lower in water (Fig. 5). At first glance this result seems unusual as water is expected to act as a plasticizer (at least at low pressure), and indeed increased hydration has been reported to decrease the value of Young’s modulus.65,72

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Fig. 5 Mechanical properties of biological assemblies. Top: normalised volume change as a function of pressure for cellulose. The solid line is a fit of the Birch-Murnaghan EOS to the data from which the bulk modulus K0 can be extracted. Bottom: the normalised shift of the inter-strand distance (4.7 Å) in TTR105-115 amyloid fibrils in water (blue) and silicone oil (red) (After Meersman et al.67 and Quesada Cabrera et al.68).

However, the presence of cavities within the fibril structure can explain this apparent contradiction. As pressure increases the cavities fill up with water, thereby making the structure more resistant to compression, whereas the much larger silicone oil molecules cannot penetrate the cavities, leaving them void and the structure remains more compressible. A similar effect has been obtained for silica glass where fast penetration by He atoms reduces the overall compressibility,73 whereas larger Ar atoms cannot significantly diffuse into the structure on the experimental timescale, leaving it more compressible (Mathieu Kint, Coralie Wiegel and Marie Foret, personal communication). The fibril cavities, however, are likely to be located outside the core of the cross-b structure as this does not dissociate at high pressure. This observation contrasts with early fibrils which have been shown to undergo dissociation under pressure.74 In this study pressure proved to be a useful tool demonstrating that amyloid fibrils undergo a maturation process during which both their packing and the fibril-stabilising interactions change.

Organisms at high pressure Life on Earth is exposed to large variations in pressure (Fig. 1). Within the oceans the hydrostatic pressure increases at a rate of

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approximately 10 MPa km1, reaching a maximum value near 100 MPa at B11 000 m depth in the Mariana Trench, the deepest point known to date.75 The average depth of the oceans is B3800 m at a pressure of 38 MPa. Almost no light reaches the deep seafloor and the temperature is normally close to 2 1C. For a long time it was thought that the deep-sea could not support life at all, but now it is recognised that the deep ocean is home to some of the richest biological environments on Earth.31,75,76 Studies began with observations of black smokers and other hydrothermal features observed at 2–5 km depth associated with undersea volcanic activity. Expeditions revealed a remarkable array of complex life forms clustering around the vents at temperatures up to 120 1C, giving rise to the concept of the ‘‘hot, deep biosphere’’.77 Micro-organisms including bacteria and archaea are found to be present in abundance from the deep-sea and in the oceanic crust both adjacent to and away from the vent sites. Some of these, such as Moritella yayanosii DB21MT-5, are obligate piezophiles, that fail to grow at atmospheric pressure.78,79 A more recent discovery is Pyrococcus CH1, an obligate piezophilic thermophile with optimal growth conditions of 52 MPa and 98 1C, recovered from a hydrothermal vent site at 4100 m depth.80 These form part of a larger class of extremophile organisms that grow under conditions of extreme pH, salinity, low and high temperature, as well as high pressure, and are found in terrestrial hydrothermal environments as well as deep inside continental crustal rocks.81 Recent evidence from one particular site, sediments from the northwestern Pacific off the Shimokita Peninsula of Japan, indicates that at least some of these micro-organisms are alive82 and that they contribute to the carbon turnover on our planet.31,83 These observational studies raise the important question of what constitutes an upper pressure limit for cells to grow, function and survive. An obligate piezophile such as M. yayanosii has optimal growth at around 80 MPa. It does not grow at pressures below 50 MPa, but still grows well at 100 MPa.79 Colwellia hadaliensis has an optimal pressure for growth of 93 MPa at 10 1C and 74 MPa at 2 1C.84 This example nicely illustrates the fact that pressure is not an independent environmental variable but that it is closely connected to the temperature, pH and salinity of the environment. This relationship is reflected in a P,T,k-phase diagram that shows the growth rate k of a given organism at any combination of pressure and temperature for given solvent conditions (Fig. 6).85 Metabolic studies of Shewanella oneidensis, a mesophilic organism belonging to the genus Shewanella that also contains piezophilic organisms and is therefore an interesting model system, have shown that these cells stop growing at B50 MPa, but maintain a slow metabolic activity up to B110 MPa.86,87 The loss of metabolic activity has been correlated with the loss of viability. These examples suggest that 120 MPa is about the upper pressure limit for microorganisms in the biosphere,80 although recent experimental results indicate that survival is possible to much higher pressures. Laboratory studies indicate that most micro-organisms are killed off at pressures between 200–600 MPa88 and this observation has led to the development and use of ‘‘pascalisation’’ as an alternative approach to thermal pasteurisation for food

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Fig. 6 Three-dimensional views of P,T,k-diagrams of three deep-sea bacterial isolates. Strain SC1 (from 1957 m) is considered piezotolerant. Strain PE36 (from 3584 m) is piezophilic at its habitat temperature. Isolate MT41 (from 10 476 m) is an obligate piezophile (After Yayanos.85).

preservation and sterilisation.89 It was therefore surprising when an American group first reported the survival of Escherichia coli MG1655 and Shewanella oneidensis at pressures up to 1600 MPa.90 The report met with some scepticism. In a critique of their observations, Yayanos commented that ‘‘Sharma et al. did not test cells in any of their experiments to determine the fraction of the cells capable of forming colonies at atmospheric pressure following exposure to high pressures.’’91 However, a subsequent study on E. coli by Vanlint et al.,92 using a directed evolution approach in which cells were ‘trained’ to survive high pressures (up to at least 2.0 GPa), provided strong evidence that cells can in fact survive such high pressures. More recent work by Aertsen and co-workers on this and other food related microorganisms, indicates that the acquisition of high pressure resistance does not occur to the same extent for all species or even for different strains of the same species.88 How do micro-organisms cope with such extreme high pressures? At the molecular level most proteins and nucleic acids are known to undergo only elastic changes in response to pressures on average up to at least 500 MPa.24 On the other hand, lipid membranes and intermolecular interactions are strongly affected at lower pressures (10–200 MPa) making these prime targets for the influence of pressure on cells. Pressure has been found to induce solubilisation of membrane proteins and permeabilisation of the outer membrane of Gram negative bacteria.93,94 This is a kinetic event that becomes irreversible at higher pressure and for longer holding times.94 Organisms living at high pressure will adapt to this condition by changing the saturated–unsaturated lipid balance in their membranes.25 A higher proportion of unsaturated lipids is also known to shift the lipid fluid–gel transition to higher temperatures.25 Pressure can also dissociate cellular assemblies such as the Ftz Z ring in bacteria and microtubules and microfilaments in eukaryotic cells.95–97 This causes morphological changes of the cell, but it has also been suggested to be responsible for growth arrest of, e.g. Lactobacillus lactis.95 Interestingly, in some deepsea organisms the Fts Z ring is found to remain intact, suggesting that some form of molecular adaptation occurs. Other targets include the interaction of aminoacyl-tRNA with

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the ribosome. Disruption of this complex due to pressureinduced conformational changes of the ribosome halts protein synthesis.98 Cells tend to counteract these changes by up-regulating the expression of stress-induced proteins. In the case of Lactobacillus sanfranciscensis DSM 20451T it was shown that the pressure-induced response led to the expression of proteins, some with possible chaperone activity, which can also be induced by other stress factors such as cold and salinity.99 Consistent with this is the finding that organisms that easily cope with oxidative or osmotic stress, such as the halophilic Halobacterium salinarum NRC-1, also display a high pressure resistance.100 Conversely, it has been shown for E. coli MG1655 that the increased pressure resistance does not confer increased heat resistance.92 These findings can be rationalised in view of the physical similarities between, on the one hand, cold and osmotic stress and pressure, and the antagonism between heat and pressure on the other hand. Nevertheless a recent study on E. coli O157 : H7 strain ATCC 43888 did report an increased cross-resistance to heat after pressure exposure.101 This was explained by the pressure-induced increase in RpoS activity, that results in an increased expression of genes involved in general stress resistance. In a follow-up study on E. coli O157 : H7 it was found that increased RpoS activity is not the only effect underlying pressure resistance.102 The conclusion so far is that there seems to be no specific pressureinduced cellular response, but that a wide range of cellular responses will be induced as a result of high pressure stress.88,99 Note that many studies have focused on mesophilic organisms that have not evolved under high pressure conditions. These studies nevertheless have potential relevance as easy acquisition of pressure resistance may pose limitations to the use of pascalisation. Descriptions of pressure adaptation and biological responses in piezophiles have been discussed elsewhere.24,103–106

Conclusions The examples given demonstrate how high hydrostatic pressure can be used to address important research questions in biochemistry and biophysics, including protein folding, enzyme reactions and the characterisation of excited states. The insight obtained from such studies often complements that from other physical and chemical perturbation methods such as heat, urea and guanidinium chloride. This is also the case in mechanical studies where high pressure enables the determination of a bulk modulus in addition to the Young’s modulus obtained from tensile experiments. Moreover, due to its particular mode of action pressure provides information on these processes and the associated energy landscape from a different perspective. This is best illustrated by the fact that pressure slows down protein dynamics and folding rates, in contrast to heat which tends to increase the reaction rates. In addition, as clearly shown in the case of excited states, one can specifically tune reactions and populations of species simply based on the volume change DV as a ‘‘selection rule’’. The ease with which

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high pressure experiments can be performed can be traced back to the important technical developments first introduced by Bridgman and developed by others continuing to the present day, many of which are adapted to biophysics and chemistry experiments under high pressure conditions. Consideration of pressure effects on biomolecules and cells is particularly relevant as a large part of the biosphere on Earth can be found in the oceans and continental crust where it is exposed to extremes of pressure and temperature. It also allows us to explore the applicability of high pressure as an alternative to pasteurisation in food processing and to create foods with novel characteristics, including texture. Controlling and harnessing the effects of the pressure variable on organisms and biomolecules can lead to new understanding of their function relevant to medical science, and they have important implications for nanotechnology applications ranging from development of structural materials and templates to nanowires and energy harvesting assemblies.68,69,107–110 The field of high pressure research constitutes an essential parameter for studying and understanding deep planetary interiors and other celestial bodies, and it is an essential tool for probing the physics and chemistry of condensed matter under extreme conditions. It has also emerged as a key technology for developing new materials with unusual structures and useful properties among inorganic systems. High pressure biophysics and chemistry studies are now emerging at a new cross-disciplinary frontier that probes and seeks to understand the conformational states and function of important macromolecular systems and complexes under conditions of extremely high density.

Acknowledgements The authors thank Professors Reinhard Lange and Wayne Hubbell, and Dr Marie-Sousai Appavou and Christian Altenbach for providing figures used to illustrate this work. Their current work in high pressure biophysical science is supported by awards from the Leverhulme Trust and the Deep Life directorate of the Deep Carbon Observatory funded by the Alfred P. Sloan Foundation.

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