Biomedical subjects

G Zaccai

Publications and source records attributed to G Zaccai.

At least 19 recordsLinked to original sources

PubMed2005

Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study.

Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.

Acetylcholinesterase↗

PubMed2005

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics.

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.

Biophysics↗

PubMed2005

Supercooled liquid-like solvent in trypsin crystals: implications for crystal annealing and temperature-controlled X-ray radiation damage studies.

The study of temperature-dependent physical changes in flash-cooled macromolecular crystals is pertinent to cryocrystallography and related issues such as crystal annealing, X-ray radiation damage and kinetic crystallography. In this context, the unit-cell volume of flash-cooled trigonal and orthorhombic trypsin crystals has been monitored upon warming from 100 to 200 K and subsequent re-cooling to 100 K. Crystals of both forms were obtained under the same crystallization conditions, yet they differ in solvent content and channel size. An abrupt non-reversible unit-cell volume decrease is observed at 185 K in orthorhombic and at 195 K in trigonal crystals as the temperature is increased; this result is consistent with ultra-viscous solvent leaving the crystals. Concomitant appearance of ice rings in the diffraction patterns suggests that the transported solvent forms crystalline ice. These results demonstrate that solvent in flash-cooled protein crystals is liquid-like near its crystallization temperature, as has been proposed, yet controversially discussed, for the case of pure water. The use of mineral oil prevents the unit-cell volume decrease in trigonal but not in orthorhombic crystals. The observation of liquid-like solvent has implications in the development of annealing protocols and points a way to the rational design of temperature-controlled crystallographic studies that aim either at studying specific radiation damage or at trapping enzymatic intermediate states.

Animals↗

PubMed2005

Proton channel hydration and dynamics of a bacteriorhodopsin triple mutant with an M-state-like conformation.

The hydration and dynamics of purple membranes (PM) containing the bacteriorhodopsin (BR) triple mutant D96G/F171C/F219L were investigated by neutron diffraction coupled with H(2)O/D(2)O exchange and by energy-resolved neutron scattering. The mutant, which is active in proton transport (Tittor et al. in J. Mol. Biol. 319:555-565, 2002), has an "open" ground-state structure similar to that of the M intermediate in the photocycle of the wild type (wt) (Subramaniam and Henderson in Nature 406:653-657, 2000). The experiments demonstrated an increased proton channel hydration in the mutant PM compared with wt PM, in both high (86%) and low (57%) relative humidity. We suggest that this is due to the smaller side chains of the mutant residues liberating space for more water molecules in the proton channel, which would then be able to participate in the proton translocation network. PM thermal dynamics has been shown to be very sensitive to membrane hydration (Lehnert et al. in Biophys. J. 75:1945-1952, 1998). The global dynamical behaviour of the mutant PM on the 100-ps time scale, as a function of relative humidity, was found to be identical to that of the wt, showing that the "open" BR structure and additional water molecules in the proton channel do not provide a softer environment enabling increased flexibility.

Bacteriorhodopsins↗

PubMed2004

The effect of water on protein dynamics.

Neutron diffraction and spectroscopy were applied to describe the hydration and dynamics of a soluble protein and a natural membrane from extreme halophilic Archaea. The quantitative dependence of protein motions on water activity was clearly illustrated, and it was established that a minimum hydration shell is required for the systems to access their functional resilience, i.e. a dynamics state that allows biological activity.

Bacteriorhodopsins↗

PubMed2004

The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study.

A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.

Biophysical Phenomena↗

PubMed2002

Tetrahedral aminopeptidase: a novel large protease complex from archaea.

A dodecameric protease complex with a tetrahedral shape (TET) was isolated from Haloarcula marismortui, a salt-loving archaeon. The 42 kDa monomers in the complex are homologous to metal-binding, bacterial aminopeptidases. TET has a broad aminopeptidase activity and can process peptides of up to 30-35 amino acids in length. TET has a central cavity that is accessible through four narrow channels (<17 A wide) and through four wider channels (21 A wide). This architecture is different from that of all the proteolytic complexes described to date that are made up by rings or barrels with a single central channel and only two openings.

Amino Acid Sequence↗

PubMed2002

Characterization of the proteasome from the extremely halophilic archaeon Haloarcula marismortui.

A 20S proteasome, comprising two subunits alpha and beta, was purified from the extreme halophilic archaeon Haloarcula marismortui, which grows only in saturated salt conditions. The three-dimensional reconstruction of the H. marismortui proteasome (Hm proteasome), obtained from negatively stained electron micrographs, is virtually identical to the structure of a thermophilic proteasome filtered to the same resolution. The stability of the Hm proteasome was found to be less salt-dependent than that of other halophilic enzymes previously described. The proteolytic activity of the Hm proteasome was investigated using the malate dehydrogenase from H. marismortui (HmMalDH) as a model substrate. The HmMalDH denatures when the salt concentration is decreased below 2 M. Under these conditions, the proteasome efficiently cleaves HmMalDH during its denaturation process, but the fully denatured HmMalDH is poorly degraded. These in vitro experiments show that, at low salt concentrations, the 20S proteasome from halophilic archaea eliminates a misfolded protein.

Archaeal Proteins↗

PubMed2001

Fast dynamics of halophilic malate dehydrogenase and BSA measured by neutron scattering under various solvent conditions influencing protein stability.

Protein thermal dynamics was evaluated by neutron scattering for halophilic malate dehydrogenase from Haloarcula marismortui (HmMalDH) and BSA under different solvent conditions. As a measure of thermal stability in each case, loss of secondary structure temperatures were determined by CD. HmMalDH requires molar salt and has different stability behavior in H(2)O, D(2)O, and in NaCl and KCl solvents. BSA remains soluble in molar NaCl. The neutron experiments provided values of mean-squared atomic fluctuations at the 0.1 ns time scale. Effective force constants, characterizing the mean resilience of the protein structure, were calculated from the variation of the mean-squared fluctuation with temperature. For HmMalDH, resilience increased progressively with increasing stability, from molar NaCl in H(2)O, via molar KCl in D(2)O, to molar NaCl in D(2)O. Surprisingly, however, the opposite was observed for BSA; its resilience is higher in H(2)O where it is less stable than in D(2)O. These results confirmed the complexity of dynamics-stability relationships in different proteins. Softer dynamics for BSA in D(2)O showed that the higher thermostability is associated with entropic fluctuations. In the halophilic protein, higher stability is associated with increased resilience showing the dominance of enthalpic terms arising from bonded interactions. From previous data, it is suggested that these are associated with hydrated ion binding stabilizing the protein in the high-salt solvent.

Animals↗

PubMed2001

Differences in the oligomeric states of the LDH-like L-MalDH from the hyperthermophilic archaea Methanococcus jannaschii and Archaeoglobus fulgidus.

L-Malate (MalDH) and L-lactate (LDH) dehydrogenases belong to the same family of NAD-dependent enzymes. To gain insight into molecular relationships within this family, we studied two hyperthermophilic (LDH-like) L-MalDH (proteins with LDH-like structure and MalDH enzymatic activity) from the archaea Archaeoglobus fulgidus (Af) and Methanococcus jannaschii (Mj). The structural parameters of these enzymes determined by neutron scattering and analytical centrifugation showed that the Af (LDH-like) L-MalDH is a dimer whereas the Mj (LDH-like) L-MalDH is a tetramer. The effects of high temperature, cofactor binding, and high phosphate concentration were studied. They did not modify the oligomeric state of either enzyme. The enzymatic activity of the dimeric Af (LDH-like) L-MalDH is controlled by a pH-dependent transition at pH 7 without dissociation of the subunits. The data were analyzed in the light of the crystallographic structure of the LDH-like L-MalDH from Haloarcula marismortui. This showed that a specific loop at the dimer-dimer contact regions in these enzymes controls the tetramer formation.

Amino Acid Sequence↗

PubMed2001

Characterization of a novel complex from halophilic archaebacteria, which displays chaperone-like activities in vitro.

We isolated a protein, P45, from the extreme halophilic archaeon Haloarcula marismortui, which displays molecular chaperone activities in vitro. P45 is a weak ATPase that assembles into a large ring-shaped oligomeric complex comprising about 10 subunits. The protein shows no significant homology to any known protein. P45 forms complexes with halophilic malate dehydrogenase during its salt-dependent denaturation/renaturation and decreases the rate of deactivation of the enzyme in an ATP-dependent manner. Compared with other halophilic proteins, the P45 complex appears to be much less dependent on salt for its various activities or stability. In vivo experiments showed that P45 accumulates when cells are exposed to a low salt environment. We suggest, therefore, that P45 could protect halophilic proteins against denaturation under conditions of cellular hyposaline stress.

Adenosine Triphosphatases↗

PubMed2001

Conformation of the Drosophila motor protein non-claret disjunctional in solution from X-ray and neutron scattering.

The quaternary structures of monomeric and dimeric Drosophila non-claret disjunctional (ncd) constructs were investigated using synchrotron x-ray and neutron solution scattering, and their low resolution shapes were restored ab initio from the scattering data. The experimental curves were further compared with those computed from crystallographic models of one monomeric and three available dimeric ncd structures in the microtubule-independent ADP-bound state. These comparisons indicate that accounting for the missing parts in the crystal structures for all these constructs is indispensable to obtain reasonable fits to the scattering patterns. A ncd construct (MC6) lacking the coiled-coil region is monomeric in solution, but the calculated scattering from the crystallographic monomer yields a poor fit to the data. A tentative configuration of the missing C-terminal residues in the form of an antiparallel beta-sheet was found that significantly improves the fit. The atomic model of a short dimeric ncd construct (MC5) without 2-fold symmetry is found to fit the data better than the symmetric models. Addition of the C-terminal residues to both head domains gives an excellent fit to the x-ray and neutron experimental data, although the orientation of the beta-sheet differs from that of the monomer. The solution structure of the long ncd construct (MC1) including complete N-terminal coiled-coil and motor domains is modeled by adding a straight coiled-coil section to the model of MC5.

Animals↗

PubMed2001

The overall conformation of conventional kinesins studied by small angle X-ray and neutron scattering.

The quaternary structures of several monomeric and dimeric kinesin constructs from Homo sapiens and Drosophila melanogaster were analyzed using small angle x-ray and neutron scattering. The experimental scattering curves of these proteins were compared with simulated scattering curves calculated from available crystallographic coordinates. These comparisons indicate that the overall conformations of the solution structures of D. melanogaster and H. sapiens kinesin heavy chain dimers are compatible with the crystal structure of dimeric kinesin from Rattus norvegicus. This suggests that the unusual asymmetric conformation of dimeric kinesin in the microtubule-independent ADP state is likely to be a general feature of the kinesin heavy chain subfamily. An intermediate length Drosophila construct (365 residues) is mostly monomeric at low protein concentration whereas at higher concentrations it is dimeric with a tendency to form higher oligomers.

Amino Acid Sequence↗

PubMed2001

Why biologists should support the exploration of Mars.

Physicists, chemists and geologists in the USA and Europe propose that the search for extraterrestrial life is an important justification for the exploration of Mars. Biologists, however, much more excited by the advent of the postgenome sequencing era, in general display little enthusiasm for planetary exploration. We argue that the search for traces of life on Mars represents a major thought-provoking challenge for the life sciences that should be taken up by the biological community.

Biology↗

PubMed2001

Biophysical characterization of the influence of salt on tetrameric SecB.

SecB is a tetrameric chaperone, with a monomeric molecular mass of 17 kDa, that is involved in protein translocation in Escherichia coli. It has been hypothesized that SecB undergoes a conformational change as a function of the salt concentration. To gain more insight into the salt-dependent behavior of SecB, we studied the protein in solution by dynamic light scattering, size exclusion chromatography, analytical ultracentrifugation, and small angle neutron scattering. The results clearly demonstrate the large influence of the salt concentration on the behavior of SecB. At high salt concentration, SecB is a non-spherical protein with a radius of gyration of 3.4 nm. At low salt concentration the hydrodynamic radius of the protein is apparently decreased, whereas the ratio of the frictional coefficients is increased. The protein solution behaves in a non-ideal way at low salt concentrations, as was shown by the analytical ultracentrifugation data and a pronounced interparticle effect observed by small angle neutron scattering. A possible explanation is a change in surface charge distribution dependent on the salt concentration in the solvent. We summarize our data in a model for the salt-dependent conformation of tetrameric SecB.

Bacterial Proteins↗

PubMed2001

Protein flexibility from the dynamical transition: a force constant analysis.

A standard analysis of the scattered neutron incoherent elastic intensity measured with very good energy resolution yields elastic scans, i.e., mean-square displacements of atomic motions (in a pico to nanosecond time scale) in a sample as a function of temperature. This provides a quick way for characterizing the dynamical behavior of biological macromolecules, such behavior being correlated with biological function and activity. Elastic scans of proteins exhibit a dynamical transition at approximately 200 K, marking a cross-over in molecular fluctuations between harmonic and nonharmonic dynamical regimes. This paper presents an approach allowing analysis of the elastic scan in terms of force constants and related parameters, such as the free energy barrier DeltaG at the transition. We find that the increased protein flexibility beyond the dynamical transition is associated with DeltaG approximately equals RT and effective force constants of the order of 0.1-3 N/m. The analysis provides a set of parameters for characterizing molecular resilience and exploring relations among dynamics, function, and activity in proteins.

Elasticity↗

PubMed2000

Moist and soft, dry and stiff: a review of neutron experiments on hydration-dynamics-activity relations in the purple membrane of Halobacterium salinarum.

Twenty-five years of neutron experiments on hydration and thermal dynamics in purple membranes of Halobacterium salinarum are reviewed. Neutron diffraction, elastic and quasielastic scattering, allowed to map the distribution of water and lipids and to measure thermal fluctuations and correlation times in the membranes, under various conditions of temperature, hydration and lipid environment. Strong correlations were established between dynamics parameters and the activity of bacteriorhodopsin (the purple membrane protein), as a light driven proton pump supporting the hypothesis that the influence of hydration on activity is in fact due to its effects on membrane thermal dynamics. Hydrogen-deuterium labelling experiments highlighted stiffer and softer parts in the bacteriorhodopsin structure. The soft parts would allow the conformational changes involved in activity, while the stiffer ones may control a valve-like function in vectorial proton transfer.

Bacteriorhodopsins↗

PubMed2000

How soft is a protein? A protein dynamics force constant measured by neutron scattering.

An effective environmental force constant is introduced to quantify the molecular resilience (or its opposite, "softness") of a protein structure and relate it to biological function and activity. Specific resilience-function relations were found in neutron-scattering experiments on purple membranes containing bacteriorhodopsin, the light-activated proton pump of halobacteria; the connection between resilience and stability is illustrated by a study of myoglobin in different environments. Important advantages of the neutron method are that it can characterize the dynamics of any type of biological sample-which need not be crystalline or monodisperse-and that it enables researchers to focus on the dynamics of specific parts of a complex structure with deuterium labeling.

Bacteriorhodopsins↗