BBA - General Subjects (v.1724, #3)

Superman's eyes by Gregory A. Petsko (225-226).

Biophysics: The end of simplicity? by Daniel Lavalette; Claude Balny (227).

Progress in analytical imaging of the cell by dynamic secondary ion mass spectrometry (SIMS microscopy) by Jean-Luc Guerquin-Kern; Ting-Di Wu; Carmen Quintana; Alain Croisy (228-238).
Keywords: Ultrastructure; Cell imaging; Ion microscopy; Dynamic secondary ion mass spectrometry; Parallel detection; Stable isotope;

Rapid identification of Candida species by FT-IR microspectroscopy by Mohammed Essendoubi; Dominique Toubas; Mohamed Bouzaggou; Jean-Michel Pinon; Michel Manfait; Ganesh D. Sockalingum (239-247).
Due to the continuous increase of human candidiasis and the great diversity of yeasts of the Candida genera, it is indispensable to identify this yeast as early as possible. Early identification enables an early diagnostic and patient-adapted anti-fungal therapy, thus reducing morbidity and mortality related to these infections. In view of this, we have in this study investigated microcolonies using a method based on Fourier transform-infrared microspectroscopy (FTIRM) for a rapid and early identification of the most frequent Candida species encountered in human pathology. FTIR spectroscopy is a whole-cell “fingerprinting” method by which microorganisms can be identified. By exploiting the huge discriminating capacity of this technique, we identified 6 species (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, Candida krusei, and Candida kefyr) from a collection of 57 clinical strains of Candida, isolated from hospitalised patients. Data obtained on 10- to 18-h-old microcolonies were compared to cultures of 24 h. Our results clearly show the efficiency and the robustness of FTIR (micro)spectroscopy in identifying species with a classification rate of 100% for both microcolonies and 24-h cultures. FTIR microspectroscopy is thus a promising clinical approach, because compared to conventional and molecular techniques, it is time and money saving, has great identification and discriminating potentials, and is amenable to an automated high-throughput routine system.
Keywords: Candida species; Early identification; Microcolony; Fourier transform-infrared (micro)spectroscopy;

New insights in the visualization of membrane permeabilization and DNA/membrane interaction of cells submitted to electric pulses by Emilie Phez; Cécile Faurie; Muriel Golzio; Justin Teissié; Marie-Pierre Rols (248-254).
Electropermeabilization designates the use of electric pulses to overcome the barrier of the cell membrane. This physical method is used to transfer anticancer drugs or genes into living cells. Its mechanism remains to be elucidated. A position-dependent modulation of the membrane potential difference is induced, leading to a transient and reversible local membrane alteration. Electropermeabilization allows a fast exchange of small hydrophilic molecules across the membrane. It occurs at the positions of the cell facing the two electrodes on an asymmetrical way. In the case of DNA transfer, a complex process is present, involving a key step of electrophoretically driven association of DNA only with the destabilized membrane facing the cathode. We report here at the membrane level, by using fluorescence microscopy, the visualization of the effect of the polarity and the orientation of electric pulses on membrane permeabilization and gene transfer. Membrane permeabilization depends on electric field orientation. Moreover, at a given electric field orientation, it becomes symmetrical for pulses of reversed polarities. The area of cell membrane where DNA interacts is increased by applying electric pulses with different orientations and polarities, leading to an increase in gene expression. Interestingly, under reversed polarity conditions, part of the DNA associated with the membrane can be removed, showing some evidence for two states of DNA in interaction with the membrane: DNA reversibly associated and DNA irreversibly inserted.
Keywords: Electric field; Electroporation; Vectoriality; Cell imaging; Membrane; DNA;

Encapsidation and transfer of phage DNA into host cells: From in vivo to single particles studies by Luc Ponchon; Stéphanie Mangenot; Pascale Boulanger; Lucienne Letellier (255-261).
A remarkable property of bacteriophages is their capacity to encapsidate large amounts of DNA during morphogenesis and to maintain their genome in the capsid in a very stable form even under extreme conditions. Even as remarkable is the efficiency with which their genome is ejected from the phage particle and transferred into the host bacteria. Biophysical techniques have led to significant progresses in characterizing these mechanisms. The molecular motor of encapsidation of several phages as well as the organization of viral capsids have been described at atomic resolution. Cryo-electron microscopy and fluorescence microscopy have permitted to describe DNA ejection at the level of single phage particles. Theoretical models of encapsidation and ejection have been proposed that can be confronted to experimental data. This review will present the state of the art on the recent advances brought by biophysics in this field. Reference will be given to the work performed on double-stranded DNA phages and on one of its representative, phage T5, our working model.
Keywords: Phage assembly; DNA encapsidation; DNA ejection; DNA packaging motor; Fluorescence microscopy;

Discriminating nevus and melanoma on paraffin-embedded skin biopsies using FTIR microspectroscopy by Ali Tfayli; Olivier Piot; Anne Durlach; Philippe Bernard; Michel Manfait (262-269).
FTIR microspectroscopy, in combination with cluster analysis, has been used to characterise skin tissues, in order to discriminate cancerous from non-cancerous ones. The main objective of this in vitro study was to demonstrate the applicability of infrared spectral imaging to separate, on paraffinised biopsies, pigmented nevi (benign skin lesions) from melanomas (malignant skin lesions). Infrared spectra were collected from paraffin-embedded samples of nevi and melanomas, without deparaffinisation. Despite the important contribution of the paraffin in these spectra, it was possible to find meaningful and discriminating spectral regions. Spectral imaging was first performed to localize different skin layers (dermis and epidermis). Spectra extracted from the images were subjected to hierarchical classification algorithm, which allowed the discrimination of melanomas from the nevi, using selected spectral windows that correspond to vibrations of DNA and melanin content. The diversity of skin lesions and direct accessibility to the skin make this organ an interesting field of investigation using this technique.
Keywords: Paraffinised tissue; Skin lesion; Melanoma; Nevus; Infrared; Microspectroscopy; Cluster analysis;

Cell electropulsation is routinely used in cell Biology for protein, RNA or DNA transfer. Its clinical applications are under development for targeted drug delivery and gene therapy. Nevertheless, the molecular mechanisms supporting the induction of permeabilizing defects in the membrane assemblies remain poorly understood. This minireview describes the present state of the investigations concerning the different steps in the reversible electropermeabilization process. The different hypotheses, which were proposed to give a molecular description of the membrane events, are critically discussed. Other possibilities are then given. The need for more basic research on the associated loss of cohesion of the membrane appears as a conclusion.
Keywords: Electropermeabilization; Electroporation; Membrane; Lipid bilayer;

Indirect evidence of submicroscopic pores in giant unilamelar vesicles by N. Rodriguez; J. Heuvingh; F. Pincet; S. Cribier (281-287).
Formation of pore-like structures in cell membranes could participate in exchange of matter between cell compartments and modify the lipid distribution between the leaflets of a bilayer. We present experiments on two model systems in which major lipid redistribution is attributed to few submicroscopic transient pores. The first kind of experiments consists in destabilizing the membrane of a giant unilamellar vesicle by inserting conic-shaped fluorescent lipids from the outer medium. The inserted lipids (10% of the vesicle lipids) should lead to membrane rupture if segregated on the outer leaflet. We show that a 5-nm diameter pore is sufficient to ease the stress on the membrane by redistributing the lipids. The second kind of experiments consists in forcing giant vesicles containing functionalized lipids to adhere. This adhesion leads to hemifusion (merging of the outer leaflets). In certain cases, the formation of pores in one of the vesicles is attested by contrast loss on this vesicle and redistribution of fluorescent labels between the leaflets. The kinetics of these phenomena is compatible with transient submicroscopic pores and long-lived membrane defects.
Keywords: Giant vesicle; Pore; Lipid translocation; Membrane destabilization; Lateral diffusion; Hemifusion; Adhesion;

A structural model of a seven-transmembrane helix receptor: The Duffy antigen/receptor for chemokine (DARC) by A.G. de Brevern; H. Wong; C. Tournamille; Y. Colin; C. Le Van Kim; C. Etchebest (288-306).
The Duffy antigen/receptor for chemokine (DARC) is an erythrocyte receptor for malaria parasites (Plasmodium vivax and Plasmodium knowlesi) and for chemokines. In contrast to other chemokine receptors, DARC is a promiscuous receptor that binds chemokines of both CC and CXC classes. The four extracellular domains (ECDs) of DARC are essential for its interaction with chemokines, whilst the first (ECD1) is sufficient for the interaction with malaria erythrocyte-binding protein. In this study, we elaborate and analyze structural models of the DARC. The construction of the 3D models is based on a comparative modeling process and on the use of many procedures to predict transmembrane segments and to detect far homologous proteins with known structures. Threading, ab initio, secondary structure and Protein Blocks approaches are used to build a very large number of models. The conformational exploration of the ECDs is performed with simulated annealing. The second and fourth ECDs are strongly constrained. On the contrary, the ECD1 is highly flexible, but seems composed of three consecutive regions: a small β-sheet, a linker region and a structured loop. The chosen structural models encompass most of the biochemical features and reflect the known experimental data. They may be used to analyze functional interaction properties.
Keywords: Malaria; Transmembrane protein; Comparative modeling; Simulated annealing; CXCL-8; Protein block;

Phosphatidylserine (PS) plays a crucial role, in the conversion of prothrombin into thrombin by the protease, factor Xa. Physiologically, the conversion occurs in the prothrombinase complex. The question of how water-soluble proteins that normally circulate in plasma bind remains to be unambiguously determined. We previously found that the amphitropic proteins (prothrombin, factors V and Va) penetrate into phospholipid layers. AC polarography has allowed the detection for the first time of insertion of factor Xa into condensed monolayers containing phosphatidylserine (PS) and phosphatidylcholine (PC) either 100% PS or 25% PS in the presence of Ca2+. This observation demonstrates that part of factor Xa can cross the phospholipid polar headgroup/hydrocarbon chain interface. In parallel experiments, radioactive surface measurements permitted measuring binding of tritium-labeled factor Xa onto a PS monolayer and calculate an association constant, 6 × 106 M−1. Penetration of factor Xa into PS-containing vesicles was investigated also using photoactivable 5-[125I]iodonaphthalene-1-azide, which binds selectively to the lipid embedded domains of the protein. These experiments suggest that Factor Xa penetrates preferentially by its heavy chain, an alternative mode of binding to the commonly accepted binding via its Gla domain. Interaction of factor Xa with PS vesicles also changes its apparent K m for S 2222.
Keywords: Factor Xa; PS; PC; Monolayers; HMDE; Vesicles;

Bicelle membranes and their use for hydrophobic peptide studies by circular dichroism and solid state NMR by Cécile Loudet; Lucie Khemtémourian; Fabien Aussenac; Stéphane Gineste; Marie-France Achard; Erick J. Dufourc (315-323).
Mixtures of dicaproyl- (DC), dimyristoyl- (DM) and 1-tetradecanoyl-2-biphenylbutanoyl-(TBB) phosphatidylcholine (PC) in water produce bicelle membranes that are oriented by magnetic fields. DMPC/DCPC systems orient such that their membrane plane is parallel to the magnetic field, whereas for TBBPC/DCPC, the plane is perpendicular to the field. Partial temperature–composition–hydration diagrams are established using solid-state 31P-NMR. DMPC/DCPC bicelles exist on a large range of composition but on a narrow temperature domain (25–45 °C). At converse, TBBPC/DCPC form bicelles on a narrow compositional range but over a large temperature span (10–70 °C). The TBBPC/DCPC bicelles are shown to be a very powerful potential tool to study the orientation of hydrophobic helices in membranes using wide line 15N-NMR. The DMPC/DCPC system that undergoes a micelle-to-bicelle transition on going from 10 °C to 40 °C may be used with circular dichroism to study the state of association of hydrophobic helices within the membrane. Results suggest that the transmembrane fragment of the neu/erbB-2 receptor is monomeric in micellar medium and dimeric/multimeric in bicelle membranes.
Keywords: Bicelle; DCPC; DMPC; TBBPC; Magnetic field orientation; neu/erbB-2 transmembrane segment; Circular dichroism; Solid State 31P and 15N NMR;

Immobilization of native membrane-bound rhodopsin on biosensor surfaces by Jasmina Minic; Jeanne Grosclaude; Josiane Aioun; Marie-Annick Persuy; Tatiana Gorojankina; Roland Salesse; Edith Pajot-Augy; Yanxia Hou; Salwa Helali; Nicole Jaffrezic-Renault; François Bessueille; Abdelhamid Errachid; Gabriel Gomila; Oscar Ruiz; Josep Samitier (324-332).
In this paper, we evaluated the grafting of G-protein-coupled receptors (GPCRs) onto functionalized surfaces, which is a primary requirement to elaborate receptor-based biosensors, or to develop novel GPCR assays. Bovine rhodopsin, a prototypical GPCR, was used in the form of receptor-enriched membrane fraction. Quantitative immobilization of the membrane-bound rhodopsin either non-specifically on a carboxylated dextran surface grafted with long alkyl groups, or specifically on a surface coated with anti-rhodopsin antibody was demonstrated by surface plasmon resonance. In addition, a new substrate based on mixed self-assembled multilayer that anchors specific anti-receptor antibodies was developed. Electrochemical impedance spectroscopy performed upon deposition of membrane-bound rhodopsin of increasing concentration exhibited a significant change, until a saturation level was reached, indicating optimum receptor immobilization on the substrate. The structures obtained with this new immobilization procedure of the rhodopsin in its native membrane environment are stable, with a controlled density of specific anchoring sites. Therefore, such receptor immobilization method is attractive for a range of applications, especially in the field of GPCR biosensors.
Keywords: G-protein-coupled receptor; Membrane; Functional immobilization; Surface plasmon resonance; Atomic force microscopy; Electrochemical impedance spectroscopy;

Study of muscle regeneration using in vitro 2D 1H spectroscopy by B. Gillet; C. Sebrié; A. Bogaert; S. Bléneau; S. de la Porte; J.-C. Beloeil (333-344).
The in vivo spectrum of regenerating muscles shows a specific cross-correlation signal assigned to the (n-3) fatty acyl chain, which peaks during the myoblast fusion phase. In order to identify the origin of this signal and to take all the lipid metabolites into account, we investigated the degeneration-regeneration process by 1H 2D NMR of lipid muscle extracts. We observed an increase in the total amount of lipids during the regeneration process, although the lipid profile did not show any drastic change during this process. The changes in the NMR signal observed in vivo and, in particular, the appearance of the specific (n-3) fatty acyl chain signal appears to arise from mobile lipid compartments located in fusing cells.
Keywords: Lipid body; Lipid extract; Myoblast; Fusion; NMR;

Divalent ion-dependent swelling of Tomato Bushy Stunt Virus: A multi-approach study by R. Aramayo; C. Mérigoux; E. Larquet; P. Bron; J. Pérez; C. Dumas; P. Vachette; N. Boisset (345-354).
Time-resolved small-angle X-ray and neutron scattering (SAXS and SANS) in solution were used to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. SAXS intensities comprise contribution from the protein capsid and the RNA moiety, while neutron scattering, recorded in 72% D2O, is essentially due to the protein capsid. Cryo-electron micrographs of compact and swollen virus were used to produce 3D reconstructions of the initial and final conformations of the virus at a resolution of 13 Å and 19 Å, respectively. While compact particles appear to be very homogeneous in size, solutions of swollen particles exhibit some size heterogeneity. A procedure has been developed to compute the SAXS pattern from the 3D reconstruction for comparison with experimental data. Cryo-electron microscopy thereby provides an invaluable starting (and ending) point for the analysis of the time-resolved swelling process using the scattering data.
Keywords: Icosahedral plant virus; Calcium ion; Structural transition; Time-resolved small-angle X-ray and neutron scattering; Cryo-electron microscopy; 3D reconstruction;

Assembly of the full-length recombinant mouse prion protein I. Formation of soluble oligomers by Charlotte Vendrely; Hélène Valadié; Lucie Bednarova; Laurent Cardin; Marielle Pasdeloup; Jéremy Cappadoro; Jan Bednar; Marguerite Rinaudo; Marc Jamin (355-366).
The conversion of a monomeric α-helix-rich isoform to multimeric β-sheet-rich isoforms is a prominent feature of the conversion between PrPC and PrPSC. We mimicked this process in vitro by exposing an unglycosylated recombinant form of the full-length mouse prion protein (MoPrP23–231) to an acidic pH, at 37 °C, and we monitored the kinetics of conformational change and assembly. In these conditions, monomeric MoPrP23–231 converts slowly to two ensembles of soluble oligomers that are separated by size exclusion chromatography. The larger oligomers (I) are unstable, and their formation involves almost no change in secondary structure content. The smaller oligomers (II) form stable spherical or annular particles containing between 8 and 15 monomers as determined by multi-angle laser light scattering (MALLS). Their formation is concomitant with the main, thought limited, change in the secondary structure content (10%) seen by Fourier Transform Infrared (FTIR) spectroscopy. Even if these oligomers conserve a large part of the secondary structure of monomeric PrP, they exhibit amyloid features with the appearance of intermolecular β-structure as revealed by the appearance of an IR band below 1620 cm−1.
Keywords: Prion protein; Protein aggregation; Protein assembly; Multi-angle laser light scattering; Fourier transform infrared spectroscopy;

Studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. We consider the surface-induced conformational changes that the recombinant ovine prion protein (ovPrP) may undergo under different pH conditions when interacting with soil minerals of highly adsorptive capacities such as montmorillonite. The conformational states of the full-length ovine prion protein adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pD range 3.5–9, using FTIR spectroscopy. The in vitro pH-induced conversion of the α-helical monomer of ovPrP into oligomers of β-like structure prone to self-aggregation does not occur when the protein is adsorbed on the clay surface. The conformation of the trapped ovPrP molecules on montmorillonite is pH-independent and looks like that of the ovPrP solvated state at pD higher than 7, suggesting the major role of Arg and Lys residues in the electrostatic origin of adsorption. The uneven distribution of positively and negatively charged residues of the ovPrP protein would promote a favored orientation of the protein towards the clay, so that not only the basic residues embedded in the N-terminal flexible part but also external basic residues in the globular part of the protein might participate to the attractive interaction. From these results, it appears unlikely that the interaction of normal prions (PrPC) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrPSc).
Keywords: Sheep prion protein; Adsorption; Conformational change; FTIR spectroscopy;

Ascididemin and meridine stabilise G-quadruplexes and inhibit telomerase in vitro by Lionel Guittat; Anne De Cian; Frédéric Rosu; Valérie Gabelica; Edwin De Pauw; Evelyne Delfourne; Jean-Louis Mergny (375-384).
Ascididemin and Meridine are two marine compounds with pyridoacridine skeletons known to exhibit interesting antitumour activities. These molecules have been reported to behave like DNA intercalators. In this study, dialysis competition assay and mass spectrometry experiments were used to determine the affinity of ascididemin and meridine for DNA structures among duplexes, triplexes, quadruplexes and single-strands. Our data confirm that ascididemin and meridine interact with DNA but also recognize triplex and quadruplex structures. These molecules exhibit a significant preference for quadruplexes over duplexes or single-strands. Meridine is a stronger quadruplex ligand and therefore a stronger telomerase inhibitor than ascididemin (IC50  = 11 and >80 μM, respectively in a standard TRAP assay).
Keywords: Telomeres; Telomerase inhibitor; G-quadruplex; G-Quartet; DNA ligands;

Molecular dynamics simulations of the unliganded T state of human hemoglobin showed the existence of a spontaneous, very wide cavity on the distal side of the α subunit. This cavity consists of three tunnels spreading from the vicinity of the iron atom (the ligand binding site) to the surface of the subunit, constituting possible passageways for the entrance of the ligand. A fourth passageway was characterized due to the trajectory of water molecules entering or leaving the heme pocket. Analogous passages were observed in the β subunits. They all appear and disappear dynamically, although some parts of them are more persistent along the trajectories. The most persistent regions within these tunnels correspond to all the xenon docking sites of human cytoglobin and to some of those of sperm whale and horse heart myoglobins and group I truncated hemoglobins.
Keywords: Human hemoglobin; Ligand migration; Xenon docking sites; Myoglobin; Cytoglobin; Group I truncated hemoglobins; Internal cavities; Molecular dynamics;

Understanding and predicting protein structures depend on the complexity and the accuracy of the models used to represent them. We have recently set up a Hidden Markov Model to optimally compress protein three-dimensional conformations into a one-dimensional series of letters of a structural alphabet. Such a model learns simultaneously the shape of representative structural letters describing the local conformation and the logic of their connections, i.e. the transition matrix between the letters. Here, we move one step further and report some evidence that such a model of protein local architecture also captures some accurate amino acid features. All the letters have specific and distinct amino acid distributions. Moreover, we show that words of amino acids can have significant propensities for some letters. Perspectives point towards the prediction of the series of letters describing the structure of a protein from its amino acid sequence.
Keywords: Hidden Markov Model; Structural alphabet; Protein structural organization; Sequence–structure relationship;

What we previously thought of as insurmountable physical and chemical barriers to life, we now see as yet another niche harbouring ‘extremophiles’. Extremophiles and their macromolecules had to develop molecular mechanisms of adaptation to extreme physico–chemical conditions. Using neutron spectroscopy, we have demonstrated that molecular dynamics represents one of these molecular mechanisms of adaptation. To which extent do hyper-saline conditions and extreme temperatures influence molecular dynamics? First, molecular dynamics were analysed for halophilic malate dehydrogenase from Haloarcula marismortui (Hm MalDH) under different molar solvent salt concentration conditions influencing its stability. Secondly, mean macromolecular motions were measured in-vivo in psychrophile (Aquaspirillum arcticum), mesophile (Escherichia coli and Proteus mirabilis), thermophile (Thermus thermophilus), and hyperthermophile (Aquifex pyrofilus) bacteria. The mean constant force of Hm MalDH increases progessively with increasing stability. The results show that the molecular adaptation of Hm MalDH to hyper-saline conditions is achieved through an increasing resilience of its structure dominated by enthalpic mechanisms. The study of bacteria has provided tools to quantify the macromolecular adaptation to extreme temperatures in the naturally crowded environment of the cell. The macromolecular resilience of bacteria increases with adaptation to high temperatures.
Keywords: Neutron scattering; Dynamics; Macromolecular adaptation; Extreme condition; Halophilic malate dehydrogenase; Bacterial adaptation;

Here, we review the dominant aspects of protein dynamics as revealed by studying hemoproteins using the combination of laser flash photolysis, kinetic spectroscopy and low temperature. The first breakthrough was the finding that geminate ligand rebinding with myoglobin is highly non-exponential at temperature T  < 200 K, providing evidence for the trapping of a large number of protein statistical substates. Another major advance was the introduction of a “model free” approach to analyze polychromatic kinetics in terms of their rate spectrum rather than to fit the data to some arbitrarily predefined kinetic scheme. Kinetic processes are identified and quantified directly from the rate spectrum without a priori assumptions. In recent years, further progresses were achieved by using xenon gas as a soft external perturbing agent that competes with ligand rebinding pathways by occupying hydrophobic protein cavities. The first part of this paper introduces several basic principles that are spread throughout a vast literature. The second part describes the main conclusions regarding conformational relaxation and ligand migration in hemoproteins obtained by combining these approaches.
Keywords: Laser photolysis; Ligand migration; Protein relaxation; Kinetics of rebinding; Hemoproteins; Protein dynamics;

New Synchrotron Radiation Circular Dichroism end-station on DISCO beamline at SOLEIL synchrotron for biomolecular analysis by Simona Miron; Matthieu Réfregiers; Anne-Marie Gilles; Jean-Claude Maurizot (425-431).
The novel Synchrotron Radiation Circular Dichroism (SRCD) technique is becoming a new tool of investigation for the molecular structures of biomolecules, like proteins, carbohydrates or others bio-materials. Here, we describe the characteristics of a new experimental end-station for circular dichroism studies, in construction on DISCO beamline at SOLEIL synchrotron (Saint-Aubin, France). This experimental end-station will be an open facility for the community of researchers in structural biology. In order to show the kind of information accessible with this type of technique, we give an example: the conformational study of the galactose mutarotase from Escherichia coli, an enzyme involved in the galactose metabolism. This study was made using an operational SRCD station available at SRS (Daresbury Laboratory, UK).
Keywords: Circular dichroism; Synchrotron Radiation Circular Dichroism; Galactose mutarotase; Protein secondary structure;

Effect of pressure on pulse radiolysis reduction of proteins by Céline Bataille; Gérard Baldacchino; Richard P. Cosson; Michèle Coppo; Catherine Trehen; Georges Vigneron; Jean Philippe Renault; Serge Pin (432-439).
Pulse radiolysis experiments were performed on proteins under pressure. Whereas many spectroscopic techniques have shown protein modifications at different pressure ranges, the present measurements performed using the water radiolysis allowed to generate radical species and to study the mechanisms implied in their reactions with proteins. This work gives the first results obtained on the effects of pressure on the rate constants of the proteins reduction by the hydrated electron at pressures up to 100 MPa. The reaction with the hydrated electron was investigated on two classes of protein: the horse myoglobin and the mussel metallothioneins. We have successively studied the influence of the pH value of metmyoglobin solutions (pH 6, 7 and 8) and the influence of the metals nature (Zn,Cu,Cd) bound to metallothioneins. For both protein, whatever the experimental conditions, the pressure does not influence the value of the reduction rate constant in the investigated range (0.1–100 MPa).
Keywords: Pressure; Pulse radiolysis; Reduction; Protein; Hydrated electron;

The pressure dependence of enzyme catalytic parameters allows volume changes associated with substrate binding and activation volumes for the chemical steps to be determined. Because catalytic constants are composite parameters, elementary volume change contributions can be calculated from the pressure differentiation of kinetic constants.Linear and non-linear pressure-dependence of single-step enzyme reactions and steady-state catalytic parameters can be observed. Non-linearity can be interpreted either in terms of interdependence between the pressure and other environmental parameters (i.e., temperature, solvent composition, pH), pressure-induced enzyme unfolding, compressibility changes and pressure-induced rate limiting changes. These different situations are illustrated with several examples.
Keywords: Hydrostatic pressure; Volume change; Activation volume; Compressibility; Rate-determining step; Inactivation; Unfolding; Dissociation;

Cumulative Contents (451-453).