Automated structure refinement for a protein heterodimer complex using limited EPR spectroscopic data and a rigid-body docking algorithm: a three-dimensional model for an ankyrin-CDB3 complex.

We report here specialized functions incorporated recently in the rigid-body docking software toolkit TagDock to utilize electron paramagnetic resonance derived (EPR-derived) interresidue distance measurements and spin-label accessibility data. The TagDock package extensions include a custom methanethiosulfonate spin label rotamer library to enable explicit, all-atom spin-label side-chain modeling and scripts to evaluate spin-label surface accessibility. These software enhancements enable us to better utilize the biophysical data routinely available from various spin-labeling experiments. To illustrate the power and utility of these tools, we report the refinement of an ankyrin:CDB3 complex model that exhibits much improved agreement with the EPR distance measurements, compared to model structures published previously.

Structural arrangement of the intracellular Ca2+ binding domains of the cardiac Na+/Ca2+ exchanger (NCX1.1): effects of Ca2+ binding.

The cardiac Na(+)/Ca(2+) exchanger (NCX1.1) serves as the primary means of Ca(2+) extrusion across the plasma membrane of cardiomyocytes after the rise in intracellular Ca(2+) during contraction. The exchanger is regulated by binding of Ca(2+) to its intracellular domain, which contains two structurally homologous Ca(2+) binding domains denoted as CBD1 and CBD2. NMR and x-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have shown how Ca(2+) binding affects their structures and motional dynamics. However, structural information on the entire Ca(2+) binding domain, denoted CBD12, and how binding of Ca(2+) alters its structure and dynamics is more limited. Site-directed spin labeling has been employed in this work to address these questions. Electron paramagnetic resonance measurements on singly labeled constructs of CBD12 have identified the regions that undergo changes in dynamics as a result of Ca(2+) binding. Double electron-electron resonance (DEER) measurements on doubly labeled constructs of CBD12 have shown that the ß-sandwich regions of the CBD1 and CBD2 domains are largely insensitive to Ca(2+) binding and that these two domains are widely separated at their N and C termini. Interdomain distances measured by DEER have been employed to construct structural models for CBD12 in the presence and absence of Ca(2+). These models show that there is not a major change in the relative orientation of the two Ca(2+) binding domains as a result of Ca(2+) binding in the NCX1.1 isoform. Additional measurements have shown that there are significant changes in the dynamics of the F-G loop region of CBD2 that merit further characterization with regard to their possible involvement in regulation of NCX1.1 activity.

Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24.

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.

Diagnosis of aged prescribed burning plumes impacting an urban area.

An unanticipated wind shift led to the advection of plumes from two prescribed burning sites that impacted Atlanta, GA, producing a heavy smoke event late in the afternoon on February 28, 2007. Observed PM2.5 concentrations increased to over 140 microg/m3 and O3 concentrations up to 30 ppb in a couple of hours, despite the late hour in February when photochemistry is less vigorous. A detailed investigation of PM2.5 chemical composition and source apportionment analysis showed that the increase in PM2.5 mass was driven mainly by organic carbon (OC). However, both results from source apportionment and an observed nonlinear relationship between OC and PM2.5 potassium (K) indicate that the increased OC was not due solely to primary emissions. Most of the OC was water-soluble organic carbon (WSOC) and was dominated by hydrophobic compounds. The data are consistent with large enhancements in isoprenoid (isoprene and monoterpenes) and other volatile organic compounds emitted from prescribed burning that led to both significant O3 and secondary organic aerosol (SOA) production. Formation of oligomers from oxidation products of isoprenoid compounds or condensation of volatile organic compounds (VOCs) with multiple functional groups emitted during prescribed burning appears to be a major component of the secondary organic contributor of the SOA. The results from this study imply that enhanced emissions due to the fire itself and elevated temperature in the burning region should be considered in air quality models (e.g., receptor and emission-based models) to assess impacts of prescribed burning emissions on ambient air quality.

Structure of the cytoplasmic domain of erythrocyte band 3 hereditary spherocytosis variant P327R: band 3 Tuscaloosa.

Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band 3 (cdb3) protein, known as band 3 Tuscaloosa, leads to a reduction in protein 4.2 content of the erythrocyte membrane and hemolytic anemia. Recent studies have shown that this point mutation does not dissociate the cdb3 dimer, nor does it lead to large-scale rearrangement of the protein structure (Bustos, S. P., and Reithmeier, R. A. F. (2006) Biochemistry 45, 1026-1034). To better define the structural changes in cdb3 that lead to the hemolytic anemia phenotype, site-directed spin labeling (SDSL), in combination with continuous wave electron paramagnetic resonance (EPR) and pulsed double electron-electron resonance (DEER) spectroscopies, has been employed in this study to compare the structure of the R327 variant with wild type P327 cdb3. It is confirmed that the P327R mutation does not dissociate the cdb3 dimer, nor does it change the spatial orientation of the two peripheral domains relative to the dimer interface. However, it does affect the packing of the C-terminal end of helix 10 of the dimerization arms in a subpopulation of cdb3 dimers, it leads to spectral changes at some residues in beta-strand 11 and in the N-terminal end of helix10, and it produces measurable spectral changes at other residues that are near the mutation site. The data indicate that the structural changes are subtle and are localized to one surface of the cdb3 dimer. The spectroscopic description of structural features of the P327R variant provides important clues about the location of one potential protein 4.2 binding surface on cdb3 as well as new insight into the structural basis of the membrane destabilization.

Mitochondrial recycling of ascorbic acid as a mechanism for regenerating cellular ascorbate.

Mitochondria are the major source of potentially damaging reactive oxygen species in most cells. Since ascorbic acid, or vitamin C, can protect against cellular oxidant stress, we studied the ability of mitochondria prepared from guinea pig skeletal muscle to recycle the vitamin from its oxidized forms. Although ascorbate concentrations in freshly prepared mitochondria were only about 0.2 mM, when provided with 6 mM succinate and 1 mM dehydroascorbate (the two-electron-oxidized form of the vitamin), mitochondria were able to generate and maintain concentrations as high as 4 mM, while releasing most of the ascorbate into the incubation medium. Mitochondrial reduction of dehydroascorbate was strongly inhibited by 1,3-bis(chloroethyl)-1-nitrosourea and by phenylarsine oxide. Despite existing evidence that mitochondrial ascorbate protects the organelle from oxidant damage, ascorbate failed to preserve mitochondrial alpha-tocopherol during prolonged incubation in oxygenated buffer. Nonetheless, the capacity for mitochondria to recycle ascorbate from its oxidized forms, measured as ascorbate-dependent ferricyanide reduction, was several-fold greater than total steady-state ascorbate concentrations. This, and the finding that more than half of the ascorbate recycled from dehydroascorbate escaped the mitochondrion, suggests that mitochondrial recycling of ascorbate might be an important mechanism for regenerating intracellular ascorbate.

Solution structure of the cytoplasmic domain of erythrocyte membrane band 3 determined by site-directed spin labeling.

The cytoplasmic domain of the anion exchange protein (cdb3) serves as a critical organizing center for protein-protein interactions that stabilize the erythrocyte membrane. The structure of the central core of cdb3, determined by X-ray crystallography from crystals grown at pH 4.8, revealed a compact dimer for residues 55-356 and unresolved N- and C-termini on each monomer [Zhang et al. (2000) Blood 96, 2925-2933]. Given that previous studies had suggested a highly asymmetric structure for cdb3 and that pH dependent structural transitions of cdb3 have been reported, the structure of cdb3 in solution at neutral pH was investigated via site-directed spin labeling in combination with conventional electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies. These studies show that the structure of the central compact dimer (residues 55-356) is indistinguishable from the crystal structure determined at pH 4.8. N-Terminal residues 1-54 and C-terminal residues 357-379 are dynamically disordered and show no indications of stable secondary structure. These results establish a structural model for cdb3 in solution at neutral pH which represents an important next step in characterizing structural details of the protein-protein interactions that stabilize the erythrocyte membrane.

Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol.

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100-1000 microM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50-100 microM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.

Reduction and uptake of methylene blue by human erythrocytes.

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB(+)) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB(+). Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB(+) <5 microM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of d-glucose. MB(+)-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB(+)-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB(+), which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB(+), that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB(+) reduction may correspond to MB(+)-dependent NAD(P)H reductase activity in erythrocyte ghosts.

Human erythrocyte recycling of ascorbic acid: relative contributions from the ascorbate free radical and dehydroascorbic acid.

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.

Mitochondrial recycling of ascorbic acid from dehydroascorbic acid: dependence on the electron transport chain.

Mitochondria can regenerate ascorbic acid from its oxidized forms, which may help to maintain the vitamin both in mitochondria and in the cytoplasm. In this work, we sought to determine the site and mechanism of mitochondrial ascorbate recycling from dehydroascorbic acid. Rat skeletal muscle mitochondria incubated for 3 h at 37 degrees C with 500 microM dehydroascorbic acid and energy substrates maintained ascorbate concentrations more than twice those observed in the absence of substrate. Succinate-dependent mitochondrial reduction of dehydroascorbic acid was blocked by inhibitors of mitochondrial Complexes II and III. Neither cytochrome c nor the outer mitochondrial membrane were necessary for the effect. The ascorbate radical was generated by mitochondria during treatment with dehydroascorbic acid and was abolished by ferricyanide, which does not penetrate the mitochondrial inner membrane. Together, these results show that energy substrate-dependent ascorbate recycling from dehydroascorbic acid involves an externally exposed portion of mitochondrial complex III.