Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na+ gradient, while the uptake of nucleobases in bacteria is powered by the H+ gradient. Here, we report the structure and function of PurTCp, a NAT family member from Colwellia psychrerythraea. The structure of PurTCp was determined to 2.80 Å resolution by X-ray crystallography. PurTCp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurTCp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H+-dependent transport of purines. Comparison of the PurTCp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.

Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, concentrative vitamin C uptake by members of the NAT family protein is driven by the Na+ gradient, while in bacteria the H+ gradient drives the uptake of nucleobases. Little is known about the mechanism of substrate binding and translocation. Here we report the structure and function of a NAT protein, PaaTCp, from the bacterium Colwellia psychrerythraea. PaaTCp transports purine bases but not pyrimidine bases, and the transport is driven by a H+ gradient. Remarkably, PaaTCp also transports vitamin C in Na+-dependent manner. The structure of PaaTCp was solved by X-ray crystallography to 2.85 Å resolution. PaaTCp forms a homodimer, and each protomer is composed of two domains, an interface domain and a transport domain. The interface domain is formed by six helices arranged in a panel-like configuration, while the transport domain is more compact with eight helices. A purine base is present in the structure and defines the location of the substrate binding site. Mutations to a conserved aspartate residue near the substrate binding site suggests that it is likely involved in mediating the H+- and Na+-dependency. Comparing the PaaTCp structure to other structures of NAT family homologs led us to propose a transport model in which conformational changes of the transport domain enable substrate translocation across the membrane.

The Kv1 family voltage-dependent K+ channels assemble with cytosolic β subunits (Kvβ), which are composed of a flexible N terminus followed by a structured core domain. The N terminus of certain Kvβs inactivates the channel by blocking the ion conduction pore, and the core domain is a functional enzyme that uses NADPH as a cofactor. Oxidation of the Kvβ-bound NADPH inhibits inactivation and potentiates channel current, but the mechanism behind this effect is unknown. Here we show that after oxidation, the core domain binds to part of the N terminus, thus restraining it from blocking the channel. The interaction is partially mediated by two negatively charged residues on the core domain and three positively charged ones on the N terminus. These results provide a molecular basis for the coupling between the cellular redox state and channel activity, and establish Kvβ as a target for pharmacological control of Kv1 channels.

The TrkH/TrkG/KtrB proteins mediate K+ uptake in bacteria and probably evolved from simple K+ channels by multiple gene duplications or fusions. Here we present the crystal structure of a TrkH from Vibrio parahaemolyticus. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to those of K+ channels but significantly shorter, is lined by backbone and side-chain oxygen atoms. Functional studies showed that TrkH is selective for permeation of K+ and Rb+ over smaller ions such as Na+ or Li+. Immediately intracellular to the selectivity filter are an intramembrane loop and an arginine residue, both highly conserved, which constrict the permeation pathway. Substituting the arginine with an alanine significantly increases the rate of K+ flux. These results reveal the molecular basis of K+ selectivity and suggest a novel gating mechanism for this large and important family of membrane transport proteins.

Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

The Shaker type voltage-dependent K+ channels (Kv1) are expressed in a wide variety of cells and essential to regulating membrane potential and cellular excitability. All Kv1 channels assemble with cytoplasmic β subunits (Kvβ) to form a stable complex. Kvβ is an aldo-keto reductase that utilizes NADPH as a cofactor, and certain Kvβs have an N-terminal segment that blocks the channel by the N-type inactivation mechanism. The enzymatic activity and channel inactivation are functionally coupled: when the Kvβ1-bound NADPH is oxidized, the N-type inactivation is inhibited and, as a result, current increases. To understand the molecular basis of the coupling, we first focused on the N-terminal segment of Kvβ1 that induces channel inactivation. We have identified a stretch of amino acid residues from the N-terminus that are required for redox modulation, which we define as the Redox Regulation Sequence (RRS). Based on our studies, we found that it is likely that the RRS binds directly to the aldo-keto reductase core of Kvβ. To test this hypothesis, and to eventually construct a mechanism for redox modulation, we have started to identify regions on the AKR core that may serve as the “receptor site” for the RRS. Initial mutational studies have identified a candidate receptor site, and structural and biochemical studies will further examine how the physical interaction is achieved, and how the interaction is dependent on the redox state.

The Shaker type voltage-dependent K+ channels (Kv1) are expressed in a wide variety of cells and essential to regulating membrane potential and cellular excitability. All Kv1 channels assemble with cytoplasmic β subunits (Kvβ) to form a macromolecule complex. Kvβ is a functional aldo-keto reductase that utilizes NADPH as cofactor, and in addition to being a functional enzyme, certain Kvβs have an N-terminal segment that blocks the channel by the N-type inactivation mechanism. The enzymatic activity and the N-type inactivation are functionally coupled: when the Kvβ-bound NADPH is oxidized, the N-type inactivation is inhibited and channel current increases as a result. Further studies showed that loss of the N-type inactivation is not due to dissociation of Kvβ upon NADPH oxidation. To understand the structural basis of the coupling mechanism, Kvβ was co-crystallized with either NADPH or NADP+, and high-resolution data sets were collected. Since NADPH is easily oxidized, for the Kvβ-NADPH complex special cares were taken to preserve the reduced cofactor throughout the crystallization process. The redox state of the cofactor was also monitored during synchrotron data collection by a micro-spectrophotometer. Results obtained from both structural analysis and functional studies led us to propose a novel mechanism of channel modulation.

The Shaker family voltage-dependent potassium channels (Kv1) assemble with cytosolic beta-subunits (Kvbeta) to form a stable complex. All Kvbeta subunits have a conserved core domain, which in one of them (Kvbeta2) is an aldoketoreductase that utilizes NADPH as a cofactor. In addition to this core, Kvbeta1 has an N terminus that closes the channel by the N-type inactivation mechanism. Point mutations in the putative catalytic site of Kvbeta1 alter the on-rate of inactivation. Whether the core of Kvbeta1 functions as an enzyme and whether its enzymatic activity affects N-type inactivation had not been explored. Here, we show that Kvbeta1 is a functional aldoketoreductase and that oxidation of the Kvbeta1-bound cofactor, either enzymatically by a substrate or non-enzymatically by hydrogen peroxide or NADP(+), induces a large increase in open channel current. The modulation is not affected by deletion of the distal C terminus of the channel, which has been suggested in structural studies to interact with Kvbeta. The rate of increase in current, which reflects NADPH oxidation, is approximately 2-fold faster at 0-mV membrane potential than at -100 mV. Thus, cofactor oxidation by Kvbeta1 is regulated by membrane potential, presumably via voltage-dependent structural changes in Kv1.1 channels.

The Shaker family voltage-dependent potassium channels (Kv1) are expressed in a wide variety of cells and are essential for cellular excitability. In humans, loss-of-function mutations of Kv1 channels lead to hyperexcitability and are directly linked to episodic ataxia and atrial fibrillation. All Kv1 channels assemble with β subunits (Kvβs), and certain Kvβs, for example Kvβ1, have an N-terminal segment that closes the channel by the N-type inactivation mechanism. In principle, dissociation of Kvβ1, although never reported, should eliminate inactivation and thus potentiate Kv1 current. We found that cortisone increases rat Kv1 channel activity by binding to Kvβ1. A crystal structure of the Kvβ-cortisone complex was solved to 1.82-Å resolution and revealed novel cortisone binding sites. Further studies demonstrated that cortisone promotes dissociation of Kvβ. The new mode of channel modulation may be explored by native or synthetic ligands to fine-tune cellular excitability.

As an alternative power source, it is very important for the nickel/metal hydride (Ni/MH) battery to improve performance. The effects of ring-metalated phthalocyanine (MPc) on inner pressure and cycling characteristics of sealed Ni/MH batteries were investigated. Experiments indicate that MPc is an effective additive against the decay of battery capacity and the pulverization of negative electrode alloy. It is advantageous to reduce the inner pressure and the inner resistance of batteries. First principle density function theory simulation revealed the activities of different MPcs for gases built up in the battery during charge and discharge cycles. It proposed that, to effectively reduce the inner pressure, an MPc should be able to activate both O-2 and H-2 and have large adsorption energy with them. This explained the observed catalytic difference among MPcs. (c) 2006 The Electrochemical Society.

The beta subunit (Kv beta) of the Shaker family voltage-dependent potassium channels (Kv1) is a cytosolic protein that forms a permanent complex with the channel. Sequence and structural conservation indicates that Kv beta resembles an aldo-keto reductase (AKR), an enzyme that catalyzes a redox reaction using an NADPH cofactor. A putative AKR in complex with a Kv channel has led to the hypothesis that intracellular redox potential may dynamically influence the excitability of a cell through Kv beta. Since the AKR function of Kv beta has never been demonstrated, a direct functional coupling between the two has not been established. We report here the identification of Kv beta substrates and the demonstration that Kv beta is a functional AKR. We have also found that channel function is modulated when the Kv beta-bound NADPH is oxidized. Further studies of the enzymatic properties of Kv beta seem to favor the role of Kv beta as a redox sensor. These results suggest that Kv beta may couple the excitability of the cell to its metabolic state and present a new avenue of research that may lead to understanding of the physiological functions of Kv beta.

The 1, 4, and 8 tyrosine (Tyr) residues on the PSII extrinsic 23 kDa protein were modified with 5, 10 or 40 mM N-acetylimidazole (NAI) respectively. The amount of rebound NAI-modified extrinsic 23 kDa protein was 98%, 80%, and 5% of that in the unmodified protein, respectively. These results indicate that the Tyr residues are absolutely essential to reconstitution ability. Further, the fluorescence and circular dichroism spectra among native and NAI-modified extrinsic 23 kDa proteins were similar, suggesting that the modification by NAI did not markedly influence the basic secondary structure of the native conformation. Thus, we have concluded that the tyrosine residues in the extrinsic 23 kDa protein are important for interaction with PSII membranes. In addition, we found that the structure of the extrinsic 23 kDa protein is stable in suspension (pH 4-9 or Tm 25-55 degrees C).

33 kD protein, located on the lumen side of thylakoid membranes, is one of three extrinsic proteins of photosystem II (PS II). Previous study showed that NBS modification of W241, the only tryptophan in 33 kD protein, is helpful for understanding the function of W241 in maintaining functional conformation of 33 kD protein. In this paper, studies of both circular dichroism and fluorescence spectra showed that upon decreasing pH from 6.2 to 2.5, the conformation of soluble 33 kD protein changed significantly, with an increase or a decrease in percentage of random coil or α-helix and turns. The changes in secondary structures of this protein are pH reversible. After NBS modification at pH 2.5, the conformational change of 33 kD protein was kept fixed. The CD ellipticity at 200 nm for NBS-modified 33 kD protein is much lower than that for control, indicating that the unfolding degree of 33 kD protein was enhanced after the NBS modification. Moreover, the conformational flexibility is lost in NBS-modified 33 kD protein, and the conformational change becomes pH irreversible, indicating that NBS modification blocked the reversibility of conformational change of 33 kD protein. The specific binding capability of NBS-modified 33 kD protein is much lower than that of low pH-treated control. Furthermore, the rebinding of modified protein on PS II membranes cannot restore the activity of oxygen evolution. We suggest that it is low pH but not NBS modification of W241 that leads to the conformational change of 33 kD protein from one functional to another non-functional state. The significant capability of proton transport of 33 kD protein is discussed.

Direct EPR evidence of the photo-generation of superoxide radicals (O2−.) was obtained by using a novel spin trapping probe in spinach Photosystem II (PS II) membrane fragments. The production of O2−. was detected by following the formation of 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) superoxide adducts (DEPMPO-OOH). The inhibition of O2−. formation by 3-(3,4-dichlorophenyl) -1,1-dimethylurea (DCMU) and the 77 K fluorescence spectrum indicated that O2−. were generated from PS II, not from PS I. The inhibition of O2−. formation by DCMU also suggested that O2−. were generated from the QBbinding site, not at a site prior to DCMU blockage. The extrinsic proteins and Mn are very important to eliminate O2−., showing that the oxygen-evolving system is involved in O2−. removal rather than production.

为了深入了解超氧自由基(O-2@)在光系统Ⅱ(PhotosystemⅡ,PSⅡ)光抑制中的作用,以5,5-二甲基-1-吡咯啉-N-氧化物(5,5-Dimethyl-1-Pyrroline-N-oxide,DMPO)为自旋捕获剂,结合EPR波谱技术,研究了PSⅡ对照和去Mn(羟胺处理)的PSⅡ颗粒在强光光抑制过程中产生O-2@随光照时间的变化.发现Mn簇的存在与否对PSⅡ光抑制产生O-2@的动态过程有很大影响,并对可能的机理进行了探讨.这些发现,对光抑制和PSⅡ结构和功能的关系研究有促进作用.

To further realize the action of Superoxide radicals (O 2 -. ) in photoinhibition of photosystem H (PS II), we employed 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap, associated with EPR spectroscopy, to study the effect of illumination time on O 2 -. formation during high light photoinhibition in PS II membranes and Mn-depleted PS II membranes. Results indicated that the removal of Mn cluster from PS II membranes has a strong influence on the dynamics of Superoxide formation. The relative mechanism was also discussed. These novel findings may further promote the studies of the structure and function of PS II and the mechanism of photoinhibition.