Structure of the Na,K-ATPase regulatory protein FXYD2b in micelles: implications for membrane-water interfacial arginines.

FXYD2 is a membrane protein responsible for regulating the function of the Na,K-ATPase in mammalian kidney epithelial cells. Here we report the structure of FXYD2b, one of two splice variants of the protein, determined by NMR spectroscopy in detergent micelles. Solid-state NMR characterization of the protein embedded in phospholipid bilayers indicates that several arginine side chains may be involved in hydrogen bond interactions with the phospholipid polar head groups. The structure and the NMR data suggest that FXYD2b could regulate the Na,K-ATPase by modulating the effective membrane surface electrostatics near the ion binding sites of the pump.

Clinical efficacy of thalidomide for various genotypes of beta thalassemia.

OBJECTIVE: The objective of this study was to investigate the therapeutic efficacy of thalidomide across various genotype presentations of ß-thalassemia so as to facilitate the early screening of thalidomide-sensitive thalassemia cases and to understand the impact of iron overload on thalidomide. METHODS: From our initial sample of 52 patients, we observed 48 patients with ß-thalassemia for two years after administration of thalidomide. This cohort included 34 patients with transfusion-dependent thalassemia (TDT) and 14 patients with non-transfusion-dependent thalassemia (NTDT). We recorded the values of hemoglobin (Hb), fetal hemoglobin (HbF), and serum ferritin (SF) in the baseline period and at 1, 3, 6, 12, 18, and 24 months after enrollment, as well as the pre- and post-treatment blood transfusion volume in all 48 cases. According to the increase in Hb levels from baseline during the 6-month observation period, the response to thalidomide was divided into four levels: main response (MaR), minor response (MiR), slow response (SLR), and no response (NR). A decrease in serum ferritin levels compared to baseline was considered alleviation of iron overload. We calculated the overall response rate (ORR) as follows: ORR = MaR + MiR + SLR/number of observed cases. RESULTS: The ORR was 91.7% (44/48 cases), and 72.9% showed MaR (35/48 cases). Among the 34 patients with TDT, 21 patients (61.8%) were free of blood transfusion, and the remaining 13 patients still required blood transfusion, but their total blood transfusion volume reduced by 31.3% when compared to the baseline. We found a total of 33 cases with 10 combinations of advantageous genes, which included 5 cases with ßCD41-42/ßCD17 and 6 cases with ßCD41-42/ß-28. Based on the treatment outcomes among the 48 cases in the observation group, there were 33 cases in the MaR group and 15 cases in the SLR/NR group. There was a difference in HbF between the two groups at baseline (P = 0.041). There were significant differences between the two groups in Hb and HbF at the time points of 6 and 12 months, respectively (P < 0.001). Compared to the baseline measurement, there was a significant decrease in the level of SF at months 12 and 24 (P < 0.001). CONCLUSION: In this study, we identified 10 ß-thalassemia gene combinations that were sensitive to thalidomide. These gene combinations can be used for initial screening and to predict the therapeutic effect of thalidomide in clinical practice. We examined the therapeutic response to thalidomide and found that the administration of thalidomide in combination with standardized iron removal was more beneficial in reducing iron overload.

[Preventive effect of ganlong capsule on chronic alcoholic hepatic injury in rats].

OBJECTIVE: To study the preventive effect of Ganlong capsule on chronic alcoholic hepatic injury in rats and its mechanism. METHOD: The rat chronic hepatic injury model was induced by intragastrically administered with gradient alcohol, once a day for 12 weeks. Efforts were made to detect the content of ALT, AST, TG, CHO, TNF-alpha in rat serum and GSH, SOD, MDA, ADH, Alb in hepatic tissues were detected, conduct a hepatic pathological examination, and pathological injury grading for livers. RESULT: Ganlong capsule could reduce the content of ALT, AST, TG in blood serum, MDA in hepatic tissues (P < 0.05), and enhance the activities of antioxidants such as SOD and GSH in hepatic tissues (P < 0.05). According to the liver histopathological observation, most structures of hepatic lobules in the model group were destroyed, with disordered liver cell cords, diffuse fat empty bubbles of different sizes in cytoplasm, focal necrosis and infiltration of inflammatory cells. All of treatment groups showed alleviation in rat liver injury to varying degrees. CONCLUSION: Ganlong capsule has a significant preventive effect to chronic alcoholic hepatic injury in rats.

Optimal decisions and Pareto improvement for green supply chain considering reciprocity and cost-sharing contract.

With the rapid development of green consumption demand, more and more consumers choose to purchase green products. Incorporating consumers' environmental awareness into a green supply chain, this paper studies the decisions and coordination of the green supply chain under the retailer's reciprocal preference. The decentralized models with and without reciprocity are constructed and analyzed with consideration of product green degree and pricing. Then, the cost-sharing joint commission contract is proposed to realize Pareto improvement. Finally, propositions and conclusions are verified by numerical simulation. The results indicate that improving consumers' environmental awareness is favorable to the profit of the whole supply chain and environment. Besides, within the reasonable range of retailer's reciprocal preference, higher value of the retailer's reciprocal preference is conductive to the better realization of environmental protection and the improvement of the economic welfare of the whole society. The cost-sharing contract exerts a positive effect in improving the environmental and economic performance in the green supply chain (GSC). The paper provides a theoretical foundation for the design of cooperative contracts in the GSC, especially the GSC with retailer's reciprocal preference.

The structure of genetically modified iron-sulfur cluster F(x) in photosystem I as determined by X-ray absorption spectroscopy.

Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, a quinone and a [4Fe-4S] iron-sulfur cluster F(X), located on the core subunits PsaA/B to iron-sulfur clusters F(A/B) on subunit PsaC. Structure function relations in the native and in the mutant (psaB-C565S/D566E) of the cysteine ligand of F(X) cluster were studied by X-ray absorption spectroscopy (EXAFS) and transient spectroscopy. The structure of F(X) was determined in PS I lacking clusters F(A/B) by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp PCC 6803. PsaC-deficient mutant cells assembled the core subunits of PS I which mediated electron transfer mostly to the phylloquinone. EXAFS analysis of the iron resolved a [4Fe-4S] cluster in the native PsaC-deficient PS I. Each iron had 4 sulfur and 3 iron atoms in the first and second shells with average Fe-S and Fe-Fe distances of 2.27 A and 2.69 A, respectively. In the C565S/D566E serine mutant, one of the irons of the cluster was ligated to three oxygen atoms with Fe-O distance of 1.81 A. The possibility that the structural changes induced an increase in the reorganization energy that consequently decreased the rate of electron transfer from the phylloquinone to F(X) is discussed.

Synergistic removal of copper and tetracycline from aqueous solution by steam-activated bamboo-derived biochar.

Steam-activated biochar (SBC) was prepared and showed excellent performance for synergistic removal of Cu2+ and tetracycline (TC). The adsorption capacity of SBC and mutual effect of TC and Cu2+ were investigated via single and binary system and the adsorption isotherm. The adsorption capacity of TC was significantly enhanced when it coexisted with Cu2+. Likewise, increased amounts of Cu2+ were adsorbed in the presence of TC. The presence of NaCl exerted a negative influence on the adsorption of Cu2+, while the inhibitory effect of salinity on TC was neutralized by bridge enhancement in the binary system. Bridge enhancement and site competition were involved in the synergistic removal of TC and Cu2+. Considering the stable application in simulated and real water samples, SBC showed great potential for synergistic removal of antibiotics and heavy metals.

Electron transfer from cytochrome c(2) to the reaction center: a transition state model for ionic strength effects due to neutral mutations.

Interprotein electron transfer plays an important role in biological energy conversion. In this work, the electron transfer reaction between cytochrome c(2) (cyt) and the reaction center (RC) was studied to determine the mechanisms coupling association and electron transfer. Previous studies have shown that mutation of hydrophobic residues in the reaction interface, particularly Tyr L162, changes the binding affinity and rates of electron transfer at low ionic strengths. In this study, the effect of ionic strength on the second-order electron transfer rate constant, k(2), between cyt c(2) and native or mutant RCs was examined. Mutations of hydrophobic and hydrogen bonding residues caused k(2) to decrease more rapidly with an increase in ionic strength. This change is explained with a transition state model by a switch from a diffusion-limited reaction in native RCs, where electron transfer occurs upon each binding event, to a fast exchange reaction in the Tyr L162 mutant, where dissociation occurs before electron transfer and k(2) depends upon the equilibrium between bound and free protein complexes. The difference in ionic strength dependence is attributed to a smaller effect of ionic strength on the energy of the transition state compared to the bound state due to larger distances between charged residues in the transition state. This model explains the faster dissociation rate at higher ionic strengths that may assist rapid turnover that is important for biological function. These results provide a quantitative model for coupling protein association with electron transfer and elucidate the role of short-range interactions in determining the rate of electron transfer.

Insights into the effect of chemical treatment on the physicochemical characteristics and adsorption behavior of pig manure-derived biochars.

Chemical treatment could improve the adsorption performance of biochars (BC). In order to deal with Pb(II) pollution, four types of biochars including unmodified, acid-treated, alkali-treated, and magnetic-treated pig manure-derived biochars (PBCs) were prepared. The effect of chemical treatment on the physical property, chemical composition, and the adsorption behavior of biochars was compared. Magnetic and alkali treatment improved pore volume and specific surface areas, and the adsorption capacity and rates were enhanced. In contrast, the adsorption capacity of acid-treated BC decreased due to the significant decrease of ash content. The magnetic samples displayed the satisfactory absorption performance, which could achieve 99.8% removal efficiency within 15 min at a Pb(II) concentration of 50 mg/L. Considering its properties of excellent adsorption performance, fast reaction rate, and convenient recovery by an external magnetic field, magnetic biochar based on pig manure may provide an effective way to remove heavy metals and decrease the pig manure solid waste.

Investigating the adsorption behavior and the relative distribution of Cd2+ sorption mechanisms on biochars by different feedstock.

The objective of this study was to investigate the adsorption behavior and the relative distribution of Cd2+ sorption mechanisms on biochars by different feedstock. Bamboo biochars (BBCs), corn straw biochars (CBCs) and pig manure biochars (PBCs) were prepared at 300-700 °C. Adsorption results showed PBCs have the best adsorption capacity for Cd2+, the extra adsorption capacity of PBCs mainly attributed to the precipitation or cation exchange, which played an important role in the removal of Cd2+ by PBCs. The contribution of involved Cd2+ removal mechanism varied with feedstock due to the different components and oxygen-containing functional groups. Cd2+-π interaction was the predominant mechanism for Cd2+ removal on biochars and the contribution proportion significantly decreased from 82.17% to 61.83% as the ash content increased from 9.40% to 58.08%. Results from this study may suggest that the application of PBC is a feasible strategy for removing metal contaminants from aqueous solutions.

Nuclear magnetic resonance structural studies of membrane proteins in micelles and bilayers.

Nuclear magnetic resonance (NMR) spectroscopy enables determination of membrane protein structures in lipid environments, such as micelles and bilayers. This chapter outlines the steps for membrane-protein structure determination using solution NMR with micelle samples, and solid-state NMR with oriented lipid-bilayer samples. The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from gamma and CHIF, two membrane proteins that function as regulatory subunits of the Na+- and K+-ATPase.

Stabilization of iron-sulfur cluster F(X) by intra-subunit interactions unraveled by suppressor and second site-directed mutations in PsaB of Photosystem I.

Intra-subunit interactions in the environment of the iron-sulfur cluster F(X) in Photosystem I (PS I) of Synechocystis sp. PCC 6803 were studied by site-directed and second site suppressor mutations. In subunit PsaB, the cysteine ligand (C565) of F(X) and a conserved aspartate (D566) adjacent to C565 were modified. The resulting mutants D566E, C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I in the thylakoids of the cyanobacterium. Yet, this is the first report of cells of the second site-suppressor mutant (D566E/L416P) and of second site-directed mutant (C565S/D566E) in PsaB that could grow autotrophically in light and were found to assemble a stable functional PS I containing all three iron-sulfur centers, F(X) and F(A/B). The newly resolved structure of PS I (PDB 1JB0) was used to interpret the functional interactions among the amino acid residues. It is suggested that the stability of F(X) is supported by a salt bridge formed between D566, which is adjacent to the cysteine ligand C565 of the iron-sulfur cluster located on loop hi, and R703 located at the start of loop jk. Hydrogen bond between R703 and D571 at the start of loop hi further stabilizes the arginine. Lengthening of the side by 1.2 A chain in mutation D566E caused destabilization of F(X). The extended side-chain was compensated for by the Fe-O, which is 0.3 A shorter than the Fe-S bond resulting in stabilization of the F(X) in the double mutations C565S/D566E. The suppressor mutation D566E/L416P allowed greater freedom for the salt bridge E566-R703, thus relieving the pressure introduced by the D566E replacement and enabling the formation of F(X). F(X) and R703 are therefore stabilized through short- and long-range interactions of the inter-helical loops between h-i, j-k and f-g, respectively.

Structures of the FXYD regulatory proteins in lipid micelles and membranes.

The FXYD membrane proteins constitute a family of conserved auxiliary subunits of the Na,K-ATPase, and have been the focus of recent attention due to their ability to finely regulate the activity of the enzyme complex in various physiological settings. In this review we describe the structures of the proteins, as well as their dynamics and their associations with the lipid bilayer membrane, which we have recently determined by NMR spectroscopy. Although the proteins are relatively small, their genes contain as many as six to nine small exons, and the coincidence of structured protein segments with their genetic elements suggests assembly from discrete structural modules through exon shuffling. The three-dimensional structures and backbone dynamics provide the foundation for understanding their intra-membrane association with the Na,K-ATPase alpha subunit, and the structure of FXYD1 suggests a mechanism whereby the phosphorylation of conserved Ser residues, by protein kinases A and C, could induce a conformational change in the cytoplasmic domain of the protein, to modulate its interaction with the alpha subunit.

NMR of membrane proteins in micelles and bilayers: the FXYD family proteins.

Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.

Control of electron transport in photosystem I by the iron-sulfur cluster FX in response to intra- and intersubunit interactions.

Photosystem I (PS I) is a transmembranal multisubunit complex that mediates light-induced electron transfer from plactocyanine to ferredoxin. The electron transfer proceeds from an excited chlorophyll a dimer (P700) through a chlorophyll a (A0), a phylloquinone (A1), and a [4Fe-4S] iron-sulfur cluster FX, all located on the core subunits PsaA and PsaB, to iron-sulfur clusters FA and FB, located on subunit PsaC. Earlier, it was attempted to determine the function of FX in the absence of FA/B mainly by chemical dissociation of subunit PsaC. However, not all PsaC subunits could be removed from the PS I preparations by this procedure without partially damaging FX. We therefore removed subunit PsaC by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp. PCC 6803. Cells could not grow under photosynthetic conditions when subunit PsaC was deleted, yet the PsaC-deficient mutant cells grew under heterotrophic conditions and assembled the core subunits of PS I in which light-induced electron transfer from P700 to A1 occurred. The photoreduction of FX was largely inhibited, as seen from direct measurement of the extent of electron transfer from A1 to FX. From the crystal structure it can be seen that the removal of subunits PsaC, PsaD, and PsaE in the PsaC-deficient mutant resulted in the braking of salt bridges between these subunits and PsaB and PsaA and the formation of a net of two negative surface charges on PsaA/B. The potential induced on FX by these surface charges is proposed to inhibit electron transport from the quinone. In the complete PS I complex, replacement of a cysteine ligand of FX by serine in site-directed mutation C565S/D566E in subunit PsaB caused an approximately 10-fold slow down of electron transfer from the quinone to FX without much affecting the extent of this electron transfer compared with wild type. Based on these and other results, we propose that FX might have a major role in controlling electron transfer through PS I.

Interactions between cytochrome c2 and photosynthetic reaction center from Rhodobacter sphaeroides: changes in binding affinity and electron transfer rate due to mutation of interfacial hydrophobic residues are strongly correlated.

The structure of the complex between cytochrome c(2) (cyt) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides shows contacts between hydrophobic residues Tyr L162, Leu M191, and Val M192 on the RC and the surface of the cyt [Axelrod et al. (2002) J. Mol. Biol. 319, 501-515]. The role of these hydrophobic residues in binding and electron transfer was investigated by replacing them with Ala and other residues. Mutations of the hydrophobic residues generally resulted in relatively small changes in the second-order electron-transfer rate k(2) (Brönsted coefficient, alpha( )()= 0.15 +/- 0.05) indicating that the transition state for association occurs before short-range hydrophobic contacts are established. Larger changes in k(2), found in some cases, were attributed to a change in the second-order mechanism from a diffusion controlled regime to a rapidly reversible binding regime. The association constant, K(A), of the cyt and the rate of electron transfer from the bound cyt, k(e), were both decreased by mutation. Replacement of Tyr L162, Leu M191, or Val M192 by Ala decreased K(A) and k(e) by factors of 130, 10, 0.6, and 120, 9, 0.6, respectively. The largest changes were obtained by mutation of Tyr L162, showing that this residue plays a key role in both binding and electron transfer. The binding affinity, K(A), and electron-transfer rate, k(e) were strongly correlated, showing that changes of hydrophobic residues affect both binding and electron transfer. This correlation suggests that changes in distance across hydrophobic interprotein contacts have similar effects on both electron tunneling and binding interactions.

Conformation of membrane-associated proapoptotic tBid.

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.