Nongeniculate coralline algae from IODP Site 310-M0005D

Material cored during the Integrated Ocean Drilling Program (IODP) Expedition 310 'Tahiti Sea Level' revealed that the fossil reef systems around Tahiti are composed of two major stratigraphic sequences: (i) a last deglacial sequence; and (ii) an older Pleistocene sequence. The older Pleistocene carbonate sequence is composed of reef deposits associated with volcaniclastic sediments and was preserved in Hole 310-M0005D drilled off Maraa. Within an approximately 70-m-thick older Pleistocene sequence (33.22-101.93 m below seafloor; 92.85-161.56 m below present sealevel) in this hole, 11 depositional units are defined by lithological changes, sedimentological features, and paleontological characteristics and are numbered sequentially from the top of the hole downward (Subunits P1-P11). Paleowater depths inferred from nongeniculate coralline algae, combined with those determined by using corals and larger foraminifers, suggest two major sealevel rises during the deposition of the older Pleistocene sequence. Of these, the second sealevel rise is associated with an intervening sealevel drop. It is likely that the second sealevel rise corresponds to that during Termination II (TII, the penultimate deglaciation, from Marine Isotope Stages 6 to 5e). Therefore, the intervening sealevel drop can be correlated with that known as the 'sealevel reversal' during TII. Because there are limited data on the Pleistocene reef systems in the tropical South Pacific Ocean, this study provides important information about Pleistocene sealevel history, the evolution of coral reef ecosystems, and the responses of coral reefs to Quaternary climate changes.

Hidden impacts of ocean acidification to live and dead coral framework

Cold-water corals, such as Lophelia pertusa, are key habitat-forming organisms found throughout the world's oceans to 3000 m deep. The complex three-dimensional framework made by these vulnerable marine ecosystems support high biodiversity and commercially important species. Given their importance, a key question is how both the living and the dead framework will fare under projected climate change. Here, we demonstrate that over 12 months L. pertusa can physiologically acclimate to increased CO2, showing sustained net calcification. However, their new skeletal structure changes and exhibits decreased crystallographic and molecular-scale bonding organization. Although physiological acclimatization was evident, we also demonstrate that there is a negative correlation between increasing CO2 levels and breaking strength of exposed framework (approx. 20-30% weaker after 12 months), meaning the exposed bases of reefs will be less effective 'load-bearers', and will become more susceptible to bioerosion and mechanical damage by 2100.

Purification and characterization of acetone carboxylase from Xanthobacter strain Py2

Acetone metabolism in the aerobic bacterium Xanthobacter strain Py2 proceeds by a carboxylation reaction forming acetoacetate as the first detectable product. In this study, acetone carboxylase, the enzyme catalyzing this reaction, has been purified to homogeneity and characterized. Acetone carboxylase was comprised of three polypeptides with molecular weights of 85,300, 78,300, and 19,600 arranged in an α2β2γ2 quaternary structure. The carboxylation of acetone was coupled to the hydrolysis of ATP and formation of 1 mol AMP and 2 mol inorganic phosphate per mol acetoacetate formed. ADP was also formed during the course of acetone consumption, but only accumulated at low, substoichiometric levels (≈10% yield) relative to acetoacetate. Inorganic pyrophosphate could not be detected as an intermediate or product of acetone carboxylation. In the absence of CO2, acetone carboxylase catalyzed the acetone-dependent hydrolysis of ATP to form both ADP and AMP, with ADP accumulating to higher levels than AMP during the course of the assays. Acetone carboxylase did not have inorganic pyrophosphatase activity. Acetone carboxylase exhibited a Vmax for acetone carboxylation of 0.225 μmol acetoacetate formed min−1⋅mg−1 at 30°C and pH 7.6 and apparent Km values of 7.80 μM (acetone), 122 μM (ATP), and 4.17 mM (CO2 plus bicarbonate). These studies reveal molecular properties of the first bacterial acetone-metabolizing enzyme to be isolated and suggest a novel mechanism of acetone carboxylation coupled to ATP hydrolysis and AMP and inorganic phosphate formation.

Two-dimensional infrared spectroscopy of peptides by phase-controlled femtosecond vibrational photon echoes

Two-dimensional infrared spectra of peptides are introduced that are the direct analogues of two- and three-pulse multiple quantum NMR. Phase matching and heterodyning are used to isolate the phase and amplitudes of the electric fields of vibrational photon echoes as a function of multiple pulse delays. Structural information is made available on the time scale of a few picoseconds. Line narrowed spectra of acyl-proline-NH2 and cross peaks implying the coupling between its amide-I modes are obtained, as are the phases of the various contributions to the signals. Solvent-sensitive structural differences are seen for the dipeptide. The methods show great promise to measure structure changes in biology on a wide range of time scales.

Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes

Na+/Cl−-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [35S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na+/Cl−-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.

Structures of two molluscan hemocyanin genes: Significance for gene evolution

We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini, were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis, corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exhibit typical signal (export) sequences, and in both cases these are interrupted by an additional intron. Each gene also contains an intron between signal peptide and FU-a and in the 3′ untranslated region. Of special relevance for evolutionary considerations are introns interrupting those regions that encode a discrete functional unit. We found that five of the eight FUs in Haliotis each are encoded by a single exon, whereas FU-f, FU-g, and FU-a are encoded by two, three and four exons, respectively. Similarly, in Octopus four of the FUs each correspond to an uninterrupted exon, whereas FU-b, FU-e, and FU-f each contain a single intron. Although the positioning of the introns between FUs is highly conserved in the two mollusks, the introns within FUs show no relationship either in location nor phase. It is proposed that the introns between FUs were generated as the eight-unit polypeptide evolved from a monomeric precursor, and that the internal introns have been added later. A hypothesis for evolution of the ring-like quaternary structure of molluscan hemocyanins is presented.

Direct conversion of an oligopeptide from a β-sheet to an α-helix: A model for amyloid formation

A 16-amino acid oligopeptide forms a stable β-sheet structure in water. In physiological solutions it is able to self-assemble to form a macroscopic matrix that stains with Congo red. On raising the temperature of the aqueous solution above 70°C, an abrupt structural transition occurs in the CD spectra from a β-sheet to a stable α-helix without a detectable random-coil intermediate. With cooling, it retained the α-helical form and took several weeks at room temperature to partially return to the β-sheet form. Slow formation of the stable β-sheet structure thus shows kinetic irreversibility. Such a formation of very stable β-sheet structures is found in the amyloid of a number of neurological diseases. This oligopeptide could be a model system for studying the protein conformational changes that occurs in scrapie or Alzheimer disease. The abrupt and direct conversion from a β-sheet to an α-helix may also be found in other processes, such as protein folding and protein–protein interaction. Furthermore, such drastic structure changes may also be exploited in biomaterials designed as sensors to detect environmental changes.

Disruption of the aldolase A tetramer into catalytically active monomers.

The fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) homotetramer has been destabilized by site-directed mutagenesis at the two different subunit interfaces. A double mutant aldolase, Q125D/E224A, sediments as two distinct species, characteristic of a slow equilibrium, with velocities expected for the monomer and tetramer. The aldolase monomer is shown to be catalytically active following isolation from sucrose density gradients. The isolated aldolase monomer had 72% of the specific activity of the wild-type enzyme and a slightly lower Michaelis constant, clearly indicating that the quaternary structure is not required for catalysis. Cross-linking of the isolated monomer confirmed that it does not rapidly reequilibrate with the tetramer following isolation. There was a substantial difference between the tetramer and monomer in their inactivation by urea. The stability toward both urea and thermal inactivation of these oligomeric variants suggests a role for the quaternary structure in maintaining the stability of aldolase, which may be an important role of quaternary structure in many proteins.