Purification and biochemical characterization of photo-active membrane protein bacteriorhodopsin from Haloarcula marismortui, an extreme halophile from the Dead Sea.

Bacteriorhodopsin (BR) is an exciting photo-active retinal protein with many potential industrial applications. In this study, BR from the extremely halophilic archaeon Haloarcula marismortui (HmBR) was purified successfully using aqueous two phase extraction method. Absorption spectroscopy analysis showed maximum absorption peak of HmBR retinal protein (λmax) at 415 nm. The purified HmBR was visualized by SDS-PAGE, with a subunit molecular mass of 27 kDa, and its identity was confirmed by resonance Raman spectroscopy, Fourier transform infrared spectroscopy and atomic force microscopy. The effect of pH and salt concentration on the absorption spectrum of HmBR was evaluated. Red-shifted in λmax of HmBR was recorded at acidic condition (pH 5) and HmBR showed remarkable optical activity under high salinity condition. The photoelectric activity of HmBR was evaluated by measuring the DC-voltage generated from HmBR coated on indium tin oxide (ITO) glass when light illumination was applied.

Potassium stress growth characteristics and energetics in the haloarchaeon Haloarcula marismortui.

Growth characteristics surrounding halophilic archaeal organisms are extremely limited in the scientific literature, with studies tending toward observing changes in cellular generation times under growth conditions limited to changes in temperature and sodium chloride concentrations. Currently, knowledge of the ionic stress experienced by haloarchaeal species through an excess or depletion of other required ions is lacking at best. The halophilic archaeon, Haloarcula marismortui, was analyzed under extreme ionic stress conditions with a specific focus on induced potassium ion stress using growth curves and analysis of the intracellular ion concentrations. Generation times were determined under potassium chloride concentrations ranging from 8 to 720 mM, and also in the presence of the alternative monovalent cations of lithium, rubidium, and cesium under limiting potassium conditions. Intracellular ion concentrations, as determined by inductively coupled mass spectrometry (ICP-MS), indicate a minimum intracellular total ion requirement of 1.13 M while tolerating up to 2.43 M intracellular concentrations. The presence of intracellular rubidium and cesium indicates that monovalent ion transport is important for energy production. Comparison of eight archaeal genomes indicates an increased diversity of potassium transport complex subunits in the halophilic organisms. Analysis of the generation times, intracellular concentrations and genome survey shows Har. marismortui exhibits an ability to cope with monovalent cation concentration changes in its native environment and provides insight into the organisms ion transport capability and specificity.

Specific cellular water dynamics observed in vivo by neutron scattering and NMR.

Neutron scattering, by using deuterium labelling, revealed how intracellular water dynamics, measured in vivo in E. coli, human red blood cells and the extreme halophile, Haloarcula marismortui, depends on the cell type and nature of the cytoplasm. The method uniquely permits the determination of motions on the molecular length (approximately ångstrøm) and time (pico- to nanosecond) scales. In the bacterial and human cells, intracellular water beyond the hydration shells of cytoplasmic macromolecules and membrane faces flows as freely as liquid water. It is not "tamed" by confinement. In contrast, in the extreme halophile archaeon, in addition to free and hydration water an intracellular water component was observed with significantly slowed down translational diffusion. The results are discussed and compared to observations in E. coli and Haloarcula marismortui by deuteron spin relaxation in NMR--a method that is sensitive to water rotational dynamics on a wide range of time scales.

Cell water dynamics on multiple time scales.

Water-biomolecule interactions have been extensively studied in dilute solutions, crystals, and rehydrated powders, but none of these model systems may capture the behavior of water in the highly organized intracellular milieu. Because of the experimental difficulty of selectively probing the structure and dynamics of water in intact cells, radically different views about the properties of cell water have proliferated. To resolve this long-standing controversy, we have measured the (2)H spin relaxation rate in living bacteria cultured in D(2)O. The relaxation data, acquired in a wide magnetic field range (0.2 mT-12 T) and analyzed in a model-independent way, reveal water dynamics on a wide range of time scales. Contradicting the view that a substantial fraction of cell water is strongly perturbed, we find that approximately 85% of cell water in Escherichia coli and in the extreme halophile Haloarcula marismortui has bulk-like dynamics. The remaining approximately 15% of cell water interacts directly with biomolecular surfaces and is motionally retarded by a factor 15 +/- 3 on average, corresponding to a rotational correlation time of 27 ps. This dynamic perturbation is three times larger than for small monomeric proteins in solution, a difference we attribute to secluded surface hydration sites in supramolecular assemblies. The relaxation data also show that a small fraction ( approximately 0.1%) of cell water exchanges from buried hydration sites on the microsecond time scale, consistent with the current understanding of protein hydration in solutions and crystals.

Neutron scattering reveals extremely slow cell water in a Dead Sea organism.

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 x 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 water-protons 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 x 10(-8) cm(2) s(-1), or 250 times lower than that of bulk water. This slow water accounts for approximately 76% 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.

The structures of four macrolide antibiotics bound to the large ribosomal subunit.

Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.

Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui.

Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo-molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of k(cat) and K(m) toward nitrate were 145 s(-1) and 79 microM, respectively, in the presence of 2.0 M NaCl.