Redox intermediates of Desulfovibrio gigas [NiFe] hydrogenase generated under hydrogen. M?ssbauer and EPR characterization of the metal centers

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and M?ssbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.     

Reconciling historical processes and population structure in the sooty tern Sterna fuscata

To test the influence of past vicariant events on population genetic structure of the sooty tern Sterna fuscata , we examined sequence variation in the mitochondrial control region of individuals from the Indo-Pacific and Atlantic Oceans. Our analyses indicate a rapid population expansion at a global scale during the last 100 000 years, consistent with global recolonisation during the interstade following the Pleistocene glacial maxima (125 000–175 000 years bp). We estimate islands of the Great Barrier Reef and Coral Sea were colonised no more than 16 000 years ago, most likely in association with the appearance of new breeding habitat following the final Pleistocene glacial retreat (19 000–22 000 years bp). Our results suggest that ice sheets linked to major glacial events not only impact genetic structuring in temperate seabirds, but that sea level changes in the tropics associated with these same events have also significantly impacted contemporary genetic structuring in tropical seabird species.     

A high affinity,calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca²⁺ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca²⁺-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca²⁺-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca²⁺ of 280 nM, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg²⁺ could replace Ca²⁺ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg²⁺ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca²⁺-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca²⁺-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca²⁺ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

Distinct regulation of salinity and genotoxic stress responses by Arabidopsis MAP kinase phosphatase 1

The Arabidopsis genome contains 20 genes encoding mitogen-activated protein kinases (MAPKs), which drastically outnumbers genes for their negative regulators, MAP kinase phosphatases (MKPs) (five at most). This contrasts sharply with genomes of other eukaryotes where the number of MAPKs and MKPs is approximately equal. MKPs may therefore play an important role in signal integration in plants, through concerted regulation of several MAPKs. Our previous studies identified Arabidopsis MKP1 and showed that its deficiency in the mkp1 mutant results in plant hypersensitivity to genotoxic stress. Here, we identify a set of MAPKs that interact with MKP1, and show that the activity level of one of these, MPK6, is regulated by MKP1 in vivo. Moreover, using expression profiling, we identified a specific group of genes that probably represent targets of MKP1 regulation. Surprisingly, the identity of these genes and interacting MAPKs suggested involvement of MKP1 in salt stress responses. Indeed, mkp1 plants have increased resistance to salinity. Thus MKP1 apparently plays a pivotal role in the integration and fine-tuning of plant responses to various environmental challenges.     

Thermal limits and adaptation in marine Antarctic ectotherms: an integrative view

A cause and effect understanding of thermal limitation and adaptation at various levels of biological organization is crucial in the elaboration of how the Antarctic climate has shaped the functional properties of extant Antarctic fauna. At the same time, this understanding requires an integrative view of how the various levels of biological organization may be intertwined. At all levels analysed, the functional specialization to permanently low temperatures implies reduced tolerance of high temperatures, as a trade-off. Maintenance of membrane fluidity, enzyme kinetic properties (Km and k(cat)) and protein structural flexibility in the cold supports metabolic flux and regulation as well as cellular functioning overall. Gene expression patterns and, even more so, loss of genetic information, especially for myoglobin (Mb) and haemoglobin (Hb) in notothenioid fishes, reflect the specialization of Antarctic organisms to a narrow range of low temperatures. The loss of Mb and Hb in icefish, together with enhanced lipid membrane densities (e.g. higher concentrations of mitochondria), becomes explicable by the exploitation of high oxygen solubility at low metabolic rates in the cold, where an enhanced fraction of oxygen supply occurs through diffusive oxygen flux. Conversely, limited oxygen supply to tissues upon warming is an early cause of functional limitation. Low standard metabolic rates may be linked to extreme stenothermy. The evolutionary forces causing low metabolic rates as a uniform character of life in Antarctic ectothermal animals may be linked to the requirement for high energetic efficiency as required to support higher organismic functioning in the cold. This requirement may result from partial compensation for the thermal limitation of growth, while other functions like hatching, development, reproduction and ageing are largely delayed. As a perspective, the integrative approach suggests that the patterns of oxygen- and capacity-limited thermal tolerance are linked, on one hand, with the capacity and design of molecules and membranes, and, on the other hand, with life-history consequences and lifestyles typically seen in the permanent cold. Future research needs to address the detailed aspects of these interrelationships.     

A de novo protein binding pair by computational design and directed evolution

The de novo design of protein-protein interfaces is a stringent test of our understanding of the principles underlying protein-protein interactions and would enable unique approaches to biological and medical challenges. Here we describe a motif-based method to computationally design protein-protein complexes with native-like interface composition and interaction density. Using this method we designed a pair of proteins, Prb and Pdar, that heterodimerize with a Kd of 130 nM, 1000-fold tighter than any previously designed de novo protein-protein complex. Directed evolution identified two point mutations that improve affinity to 180 pM. Crystal structures of an affinity-matured complex reveal binding is entirely through the designed interface residues. Surprisingly, in the in vitro evolved complex one of the partners is rotated 180° relative to the original design model, yet still maintains the central computationally designed hotspot interaction and preserves the character of many peripheral interactions. This work demonstrates that high-affinity protein interfaces can be created by designing complementary interaction surfaces on two noninteracting partners and underscores remaining challenges.     

Simplified enrichment of plasma membrane proteins for proteomic analyses in Arabidopsis thaliana

The plasma membrane (PM) serves as the point of contact between cells and the outside environment. As such, changes in the PM proteome are an important component of understanding cellular responses to a diverse array of stimuli. However, intricate sample handling to enrich PM proteomes by traditional methods is both technically challenging and time consuming. Here, we describe a simplified method for decreasing the representation of other membrane-containing organelles such as the endoplasmic reticulum, plastids and mitochondria from crude microsomal membrane isolations. The decrease in other organellar proteomes results in an increase in both the total number of PM proteins and the number of spectra identified from these proteins representing the PM proteome. Therefore, this strategy represents a simple and rapid method for enriching PM proteins from Arabidopsis cell cultures for proteomic analyses.