Mean macromolecular dynamics was quantified in vivo by neutron scattering in psychrophile, mesophile, thermophile and hyperthermophile bacteria. Root mean square atomic fluctuation amplitudes determining macromolecular flexibility were found to be similar for each organism at its physiological temperature (B1 Å in the 0.1 ns timescale). Effective force constants determining the mean macromolecular resilience were found to increase with physiological temperature from 0.2 N/m for the psychrophiles, which grow at 4 1C, to 0.6 N/m for the hyperthermophiles (85 1C), indicating that the increase in stabilization free energy is dominated by enthalpic rather than entropic terms. Larger resilience allows macromolecular stability at high temperatures, while maintaining flexibility within acceptable limits for biological activity.

Intracellular water dynamics in Haloarcula marismortui, an extremely halophilic organism originally isolated from the Dead Sea, was studied by neutron scattering. The water in centrifuged cell pellets was examined by means of two spectrometers, IN6 and IN16, sensitive to motions with time scales of 10 ps and 1 ns, respectively. From IN6 data, a translational diffusion constant of 1.3 ؋ 10 ؊5 cm 2 s ؊1 was determined at 285 K. This value is close to that found previously for other cells and close to that for bulk water, as well as that of the water in the 3.5 M NaCl solution bathing the cells. A very slow water component was discovered from the IN16 data. At 285 K the waterprotons of this component displays a residence time of 411 ps (compared with a few ps in bulk water). At 300 K, the residence time dropped to 243 ps and was associated with a translational diffusion of 9.3 ؋ 10 ؊8 cm 2 s ؊1 , or 250 times lower than that of bulk water. This slow water accounts for Ϸ76% of cell water in H. marismortui. No such water was found in Escherichia coli measured on BSS, a neutron spectrometer with properties similar to those of IN16. It is hypothesized that the slow mobility of a large part of H. marismortui cell water indicates a specific water structure responsible for the large amounts of K ؉ bound within these extremophile cells.Haloarcula marismortui ͉ water structure and dynamics ͉ extreme haplophile T he study of the specific properties of water in biological systems continues to yield fascinating surprises. Haloarcula marismortui, an archaeal extreme halophile isolated from the Dead Sea, attracted our attention some years ago because of its high selectivity for, despite a high membrane permeability (1). In distinction to other organisms, K ϩ was retained within the cell, even in the absence of metabolism, with a half-time of exchange with the outside medium of Ͼ24 h (2). At the time, the only systems known with a very high binding selectivity were antibiotics such as nonactin and valinomycin. The structure responsible for their specificity is a ''cage'' of six to eight carbonyl oxygen atoms arranged in space according to a definite pattern. If the same principle were to be responsible for K ϩ binding in H. marismortui, 24-32 moles oxygen per liter of cell water would be required, enormous amounts of oxygen atoms that cannot be supplied exclusively by the organic components of the cell. It is therefore suggested that the oxygen atoms required might be furnished by the ordering of water molecules forming a tertiary system of water, KCl, and cell proteins.Evidence in favor of more than a single phase of water in cell pellets of H. marismortui came initially from H-NMR measurements (3). The visible intensity of water in such pellets accounted for all of the total water present (101 Ϯ 7%). When the pellets were slowly cooled to below Ϫ19°C (the temperature at which the culture medium froze), 40% of the water signal remained visible. From analysis of the dynamic properties of the intracellular water, it was concluded ...

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.

International audienceSometimes less is more: [13C1H3]methyl isotopomers can be biosynthetically incorporated specifically into the pro-S methyl groups of leucine and valine residues in large protein assemblies within a perdeuterated background by using an acetolactate precursor. This stereospecific labeling strategy considerably enhances NMR spectra for large protein assemblies

To explore protein adaptation to extremely high temperatures, two parameters related to macromolecular dynamics, the mean square atomic fluctuation and structural resilience, expressed as a mean force constant, were measured by neutron scattering for hyperthermophilic malate dehydrogenase from Methanococcus jannaschii and a mesophilic homologue, lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle. The root mean square fluctuations, defining flexibility, were found to be similar for both enzymes (1.5 Å) at their optimal activity temperature. Resilience values, defining structural rigidity, are higher by an order of magnitude for the high temperature-adapted protein (0.15 Newtons/meter for O. cunniculus lactate dehydrogenase and 1.5 Newtons/meter for M. jannaschii malate dehydrogenase). Thermoadaptation appears to have been achieved by evolution through selection of appropriate structural rigidity in order to preserve specific protein structure while allowing the conformational flexibility required for activity.Hyperthermophilic organisms grow at temperatures above 80°C. Proteins from these organisms are themselves optimally active between 60 and 125°C and serve as paradigms for the characterization of factors responsible for protein fold stability and flexibility. Hyperthermophilic enzymes have also attracted considerable attention because of a range of biotechnological applications (1, 2).Sequence comparison studies and structural analyses carried out on hyperthermophilic proteins and their mesophilic homologues have shown that thermal stability is associated with multiple factors, including an increase in hydrogen bonding, complex salt bridge formation, and helix stabilization by acidic residues. The commonly accepted hypothesis is that increased thermal stability is due to enhanced conformational rigidity of the molecular structure (3). Hyperthermophilic enzymes are also characterized by a higher temperature of maximum activity (3, 4). The more rigid hyperthermophilic enzyme would then require higher temperatures to achieve the requisite conformational flexibility for activity.Experiments have shown that thermostable enzymes exhibit reduced structural flexibility at room temperature with respect to their mesophilic homologues (4, 5), whereas others, on ␣-amylase (6) and on rubredoxin (7,8), have shown the opposite effect, i.e. the thermostable homologues were found to be more flexible, suggesting stabilization through entropic effects. Relations between flexibility and stability are, therefore, complex. Atomic fluctuations only were measured in these experiments and interpreted in terms of flexibility.It is important to point out that reduced structural "flexibility" does not necessarily imply a more "rigid" structure. Atoms are maintained in a structure by forces that link them to their neighbors. In terms of a force field, the width of the potential well in which an atom moves is a measure of its flexibility in terms of a root mean square fluctuation amplitude ͑ ͌ Ͻu 2 Ͼ), whereas the detailed ...

Prokaryotes inhabiting in the deep sea vent ecosystem will thus experience harsh conditions of temperature, pH, salinity or high hydrostatic pressure (HHP) stress. Among the fifty-two piezophilic and piezotolerant prokaryotes isolated so far from different deep-sea environments, only fifteen (four Bacteria and eleven Archaea) that are true hyper/thermophiles and piezophiles have been isolated from deep-sea hydrothermal vents; these belong mainly to the Thermococcales order. Different strategies are used by microorganisms to thrive in deep-sea hydrothermal vents in which "extreme" physico-chemical conditions prevail and where non-adapted organisms cannot live, or even survive. HHP is known to impact the structure of several cellular components and functions, such as membrane fluidity, protein activity and structure. Physically the impact of pressure resembles a lowering of temperature, since it reinforces the structure of certain molecules, such as membrane lipids, and an increase in temperature, since it will also destabilize other structures, such as proteins. However, universal molecular signatures of HHP adaptation are not yet known and are still to be deciphered.

Obtaining sequence-specific assignments remains a major bottleneck in solution NMR investigations of supramolecular structure, dynamics and interactions. Here we demonstrate that resonance assignment of methyl probes in high molecular weight protein assemblies can be efficiently achieved by combining fast NMR experiments, residue-type-specific isotope-labeling and automated site-directed mutagenesis. The utility of this general and straightforward strategy is demonstrated through the characterization of intermolecular interactions involving a 468-kDa multimeric aminopeptidase, PhTET2.

Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.