Role In Energy Coupling Research Articles

Validation of magnetized gas-jet experiments to investigate the effects of an external magnetic field on laser-plasma instabilities

Laser-plasma instabilities (LPI) play a detrimental role in energy coupling to the target in inertial confinement fusion (ICF). The recent development of applied strong magnetic fields for use in ICF and laboratory astrophysics experiments has opened opportunities to investigate the role of external magnetic fields on LPIs. Recent numerical studies have shown that stimulated Raman scattering (SRS) can be mitigated by external magnetic fields in the kinetic regime of the instability and warrant systematic experimental studies to validate modelling. To this end, we design experiments at the OMEGA-EP laser facility to investigate the effect of an external perpendicular $B$ -field of 5–30 T on the backscattered light spectrum from a gas-jet target. We present measurements from a proof-of-principle experiment, where the backward-SRS (BSRS) is in the kinetic regime, for which the magnetization is expected to produce the greatest effects on instability growth. New simulations motivated by this experiment are used to inform the proposal of an upgraded experimental design. Our simulation predictions show that the new design is suited to experimentally demonstrating BSRS mitigation by an external magnetic field, despite the magnetization effects on the hydrodynamics, as well as the inherent temporal and spatial variations in plasma conditions.

Crystal structure of a folate energy-coupling factor transporter from Lactobacillus brevis

ATP-binding cassette (ABC) transporters, composed of importers and exporters, form one of the biggest protein superfamilies that transport a variety of substrates across the membrane, powered by ATP hydrolysis. Most ABC transporters are composed of two transmembrane domains and two cytoplasmic nucleotide-binding domains. Also, importers from prokaryotes usually have extra solute-binding proteins in the periplasm that are responsible for the binding of substrates. Structures of importers have been reported that suggested a two-state model for the transport mechanism. Energy-coupling factor (ECF) transporters belong to a new class of ATP-binding cassette importers. Each ECF transporter comprises an energy-coupling module consisting of a transmembrane T protein (EcfT), two nucleotide-binding proteins (EcfA and EcfA'), and another transmembrane substrate-specific binding S protein (EcfS). Despite the similarities with ABC transporters, ECF transporters have different organizational and functional properties. The lack of solute-binding proteins in ECF transporters differentiates them clearly from the canonical ABC importers. Previously reported structures of the EcfS proteins RibU and ThiT clearly demonstrated the binding site of substrate riboflavin and thiamine, respectively. However, the organization of the four different components and the transport mechanism of ECF transporters remain unknown. Here we present the structure of an intact folate ECF transporter from Lactobacillus brevis at a resolution of 3 Å. This structure was captured in an inward-facing, nucleotide-free conformation with no bound substrate. The folate-binding protein FolT is nearly parallel to the membrane and is bound almost entirely by EcfT, which adopts an L shape and connects to EcfA and EcfA' through two coupling helices. Two conserved XRX motifs from the coupling helices of EcfT have a vital role in energy coupling by docking into EcfA-EcfA'. We propose a transport model that involves a substantial conformational change of FolT.

The Secondary Multidrug/Proton Antiporter MdfA Tolerates Displacements of an Essential Negatively Charged Side Chain

The largest family of solute transporters includes ion motive force-driven secondary transporters. Several well characterized solute-specific transport systems in this group have at least one irreplaceable acidic residue that plays a critical role in energy coupling during transport. Previous studies have established the importance of acidic residues in substrate recognition by major facilitator superfamily secondary multidrug transporters, but their role in the transport mechanism remained unknown. We have been investigating the involvement of acidic residues in the mechanism of MdfA, an Escherichia coli secondary multidrug/proton antiporter. We demonstrated that no single negatively charged side chain plays an irreplaceable role in MdfA. Accordingly, we hypothesized that MdfA might be able to utilize at least two acidic residues alternatively. In this study, we present evidence that indeed, unlike solute-specific secondary transporters, MdfA tolerates displacements of an essential negative charge to various locations in the putative drug translocation pathway. The results suggest that MdfA utilizes a proton translocation strategy that is less sensitive to perturbations in the geometry of the proton-binding site, further illustrating the exceptional structural promiscuity of multidrug transporters.

The arginine finger of the Bloom syndrome protein: its structural organization and its role in energy coupling

RecQ family helicases are essential in maintaining chromosomal DNA stability and integrity. Despite extensive studies, the mechanisms of these enzymes are still poorly understood. Crystal structures of many helicases reveal a highly conserved arginine residue located near the γ-phosphate of ATP. This residue is widely recognized as an arginine finger, and may sense ATP binding and hydrolysis, and transmit conformational changes. We investigated the existence and role of the arginine finger in the Bloom syndrome protein (BLM), a RecQ family helicase, in ATP hydrolysis and energy coupling. Our studies by combination of structural modelling, site-directed mutagenesis and biochemical and biophysical approaches, demonstrate that mutations of residues interacting with the γ-phosphate of ATP or surrounding the ATP-binding sites result in severe impairment in the ATPase activity of BLM. These mutations also impair BLM's DNA-unwinding activities, but do not affect its ATP and DNA-binding abilities. These data allow us to identify R982 as the residue that functions as a BLM arginine finger. Our findings further indicate how the arginine finger is precisely positioned by the conserved motifs with respect to the γ-phosphate.

The arginine finger of bacteriophage T7 gene 4 helicase: role in energy coupling.

The DNA helicase encoded by gene 4 of bacteriophage T7 couples DNA unwinding to the hydrolysis of dTTP. The loss of coupling in the presence of orthovanadate (Vi) suggests that the gamma-phosphate of dTTP plays an important role in this mechanism. The crystal structure of the hexameric helicase shows Arg-522, located at the subunit interface, positioned to interact with the gamma-phosphate of bound nucleoside 5' triphosphate. In this respect, it is analogous to arginine fingers found in other nucleotide-hydrolyzing enzymes. When Arg-522 is replaced with alanine (gp4-R522A) or lysine (gp4-R522K), the rate of dTTP hydrolysis is significantly decreased. dTTPase activity of the altered proteins is not inhibited by Vi, suggesting the loss of an interaction between Vi and gene 4 protein. gp4-R522A cannot unwind DNA, whereas gp4-R522K does so, proportionate to its dTTPase activity. However, gp4-R522K cannot stimulate T7 polymerase activity on double-stranded DNA. These findings support the involvement of the Arg-522 residue in the energy coupling mechanism.

Facilitated Drug Influx by an Energy-uncoupled Secondary Multidrug Transporter

The majority of bacterial multidrug resistance transporters belong to the class of secondary transporters. LmrP is a proton/drug antiporter of Lactococcus lactis that extrudes positively charged lipophilic substrates from the inner leaflet of the membrane to the external medium. This study shows that LmrP is a true secondary transporter. In the absence of a proton motive force, LmrP facilitates downhill fluxes of ethidium in both directions. These fluxes are inhibited by other substrates of LmrP. The cysteine-reactive agent p-chloromercuri-benzene sulfonate inhibits these fluxes in wild type LmrP but not in the cysteine-less LmrP C270A mutant. Cysteine mutagenesis of LmrP resulted in three mutants, D68C/C270A, D128C/C270A, and E327C/C270A, with an energy-uncoupled phenotype. Asp68 is located in the conserved motif GXXX(D/E)(R/K)XGRK for the major facilitator superfamily of secondary transporters and was found to play an important role in energy coupling, whereas the negatively charged residues Asp128 and Glu327 have indirect effects on the transport process. L. lactis strains expressing these uncoupled mutants of LmrP show an increased rate of ethidium influx and an increased drug susceptibility compared with cells harboring an empty vector. The rate of influx in these mutants is enhanced by a transmembrane electrical potential, inside negative. These observations suggest a new strategy for eliminating drug-resistant microbial pathogens, i.e. the design and use of modulators of secondary multidrug resistance transporters that uncouple drug efflux from proton influx, thereby allowing transmembrane electrical potential-driven influx of cationic drugs.

New functions for coenzyme Q

Coenzyme Q is primarily identified with its role in energy coupling where it is involved in the generation of a proton gradient across membranes to drive ATP formation. Its identification as a significant antioxidant throughout cellular membranes is developing. Its function in other membrane redox systems introduces new functions such as the generation of hydrogen peroxide related to cellular signal systems or the acidification of other organelles. A role in the control of cell growth and apoptosis has also been introduced.

Histidine-419 plays a role in energy coupling in the vesicular monoamine transporter from rat

Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. It has been suggested in the past that His residues play a role in H + movement or in its coupling to active transport in H +-symporters and antiporters. Indeed VMAT-mediated transport is inhibited by reagents specific for His residues. We have identified one His residue in VMAT1 from rat which is conserved in other vesicular neurotransmitter transporters. Mutagenesis of this His (H419) to either Arg or Cys completely inhibits [ 3H]serotonin and [ 3H]dopamine accumulation. Mutagenesis also inhibits other H +-dependent partial reactions of VMAT such as the acceleration of binding of the high affinity ligand reserpine, but does not inhibit the [ 3H]reserpine binding which is not dependent on H + translocation. It is concluded that His-419 plays a role in energy coupling in r-VMAT1.

6] Rate of redox reactions related to surface potential and other surface-related parameters in biological membranes

Publisher Summary This chapter discusses the rate of redox reactions related to surface potential and other surface-related parameters in biological membranes. The generation and use of the electrochemical free energy of protons or other ions play a crucial role in energy coupling between electron transfer, adenosine triphosphate (ATP) synthesis, and ion transport, performed on the membranes according to the chemiosmotic theory. The surfaces of biological membranes are at a negative electrical potential with respect to the bulk aqueous phase in the physiological pH range because of the charges immobilized on lipid and protein components. The interchange between the electrical and chemical terms introduces significant effects on the reactions of the membrane components introducing electrical and chemical environments different from those in the bulk phase. Effects of these phenomena on the energy transducing reactions are discussed in this chapter.

Protonic and cationic changes during cyclical cation transport driven by electron transfer

Complexes I and III in mitochondrial particles mediate the coupling of electron transfer (NADH → Q; QH 2 → ferricytochrome c ) to either cyclical or net transport of K +. Cyclical transport was characterized by a U-shaped profile for the protonic and cationic changes observed during an oxygen pulse. The K + e and H + e ratios were found to be unity for the coupled reactions mediated by Complexes I and III. Protons were released on the side of the inner membrane where oxidation was initiated (I side) and taken up on the side where oxidation was terminated (M side). The pattern now shown to be general for the three electron transfer complexes that collectively carry out the NADH → O 2 sequence includes dehydrogenation of the primary reductants on the I side, hydrogenation of the terminal acceptors on the M side, and nonergized transfer of a proton from the I to the M side of the membrane by cation/proton exchange. From these data it has been concluded that the movements of the electron and cation are coupled and synchronized, whereas the proton plays no direct role in energy coupling mediated by electron transfer complexes.

The entry process as the target for energy input in active transport of alpha-aminoisobutyric acid by Mycobacterium phlei.

Mycobacterium phlei was shown to accumulate alpha-aminoisobutyric acid, establishing a concentration gradient of approximately 15,000-fold. The apparent affinity constant of the carrier for alpha-aminoisobutyric acid was 1.8 microM. The system exhibited a broad specificity provided two structural requirements were satisfied: the presence of a free amino and carboxyl group on the alpha carbon and the absence of a net charge. The role of energy coupling on the accumulation of alpha-aminoisobutyric acid was studied by two different kinds of experiments, the relative effects of the inhibitors on the rate of entry and the steady-state of accumulation, and a comparison of the efflux induced at the final steady state by the addition of (a) excess nonradioactive alpha-aminoisobutyric acid, (b) energy inhibitors, or (c) both. The results are consistent with the hypothesis that accumulation of alpha-aminoisobutyric acid is due to an increased rate of entry, the rate of exit not being affected by metabolic inhibitors.

Existence of electrogenic hydrogen ion/sodium ion antiport in Halobacterium halobium cell envelope vesicles.

Illumination causes the extrusion of protons from Halobacterium halobium cell envelope vesicles, as a result of the action of light on bacteriorhodopsin. The protonmotive force developed is coupled to the active transport of Na+ out of the vesicles. The light-dependent ion fluxes in these vesicles were studied by following changes in the external pH, in the fluorescence of the dye, 3,3'-dipentyloxadicarbocyanine, in the 22Na content of the vesicles, and in [3H]dibenzyldimethylammonium (DDA+) accumulation. During Na+ efflux, and dependent on the presence of Na+ inside the vesicles, the initial light-induced H+ extrusion is followed by H+ influx, which results in net alkalinization of the medium at pH greater than 6.5. When the Na+ content of the vesicles is depleted, the original net of the medium is restored and large deltapH develops, accompanied by a decrease in the electrical potential. Data reported elsewhere suggest that the driving force for the transport of some amino acids consists mainly of the electrical potential, while for others it comprises the Na+ gradient as well. Glutamate transport appears to be energized only by the Na+ gradient. The development of the Na+ gradient during illumination thus plays an important role in energy coupling. The results obtained are consistent with the existence of an electrogenic H+/Na+ antiport mechanism (H+/Na+ greater than 1) in H halobium which facilitates the uphill Na+ efflux. The light-induced protonmotive force thereby becomes the driving force in forming a Na+ gradient. The presence of the proposed H+/Na+ antiporter explains many of the light-induced pH effects in intact H. halobium cells.

Functions of a new photoreceptor membrane.

The purple membrane of Halobacterium halobium contains only one protein, bacteriorhodopsin, which closely resembles the visual pigments of animals. Light flashes cause a rapid transient shift of its absorption maximum from 560 to 415 nm. This shift is accompanied by release and uptake of protons. Respiring cells acidify the medium in the dark; if they contain purple membrane their O(2) consumption is reduced in the light. Starved or anaerobic cells containing purple membrane, in the absence of any apparent source of energy, generate and maintain a proton gradient across the cell membrane as long as they are exposed to light. We postulate that the light-generated proton gradient arises from a vectorial release and uptake of protons by bacteriorhodopsin, which is suitably oriented in the cell membrane and under continuous illumination oscillates rapidly between the long- and short-wavelength form. Preliminary results indicate that the gradient in H. halobium plays the central role in energy coupling attributed to such electrochemical gradients by Mitchell's chemiosmotic theory.

Accumulation of Neutral Amino Acids by Streptococcus faecalis: ENERGY COUPLING BY A PROTON-MOTIVE FORCE

Abstract We have studied the accumulation of amino acids by a strain of Streptococcus faecalis (faecium) which does not carry out oxidative phosphorylation but relies upon glycolysis for metabolic energy. In the absence of exogenous substrate, cells take up limited amounts of glycine, l-alanine, l-serine, and l-threonine by exchange for components of the free amino acid pool. Net flux is much slower. When an energy source is provided, extensive net uptake takes place. Glycine and threonine accumulate largely as such, attaining concentration gradients of about 400 between cytoplasm and medium. Glycine, alanine, serine, and threonine undergo reciprocal exchange in starving cells, suggesting that they share a common transport system. A mutant, isolated by virtue of its resistance to cycloserine, was severely deficient in the transport of all four amino acids, both in the presence and in absence of an energy source. It thus appears that there is a single transport system for these amino acids, which can operate in two modes: tightly coupled exchange which is independent of metabolism, and net uptake linked to energy generation. The energy donor for metabolic accumulation is most probably ATP. Inhibition of accumulation by dicyclohexylcarbodiimide further implicates the membrane-bound ATPase in coupling glycolysis to the transport system. No evidence for an obligatory involvement of either K+ or Na+ was obtained, but inhibition by uncouplers strongly suggests that protons play a key role in energy coupling of amino acid transport. The effects of ionophores on amino acid accumulation are best understood in terms of Mitchell's chemiosmotic hypothesis: extrusion of protons by the ATPase would generate a gradient of pH and of electrical potential (proton-motive force) across the membrane and establish a circulation of protons which is the immediate driving force for active transport. Metabolic accumulation (unlike exchange in starving cells) was totally blocked by proton-conducting uncouplers. These also induced release of glycine and threonine previously accumulated by the cells. Nigericin also inhibited accumulation and elicited release, but valinomycin and monactin were severely inhibitory only at K+ concentrations comparable to those in the cytoplasm. These and other results suggest that the membrane potential is the primary component of the proton-motive force. A pH gradient is not obligatory, but an alkaline cytoplasm may well be necessary. Accumulation of threonine and glycine could be elicited even in starving cells, by artificially imposing an electrical potential across the membrane. This was done by addition of valinomycin to K+-loaded cells suspended in a medium free of K+, so as to induce K+ efflux. Accumulation under these conditions did not involve ATP or the ATPase, but was sensitive to proton-conducting uncouplers. We believe that the electrical potential supports accumulation by co-transport of the substrate with protons, as envisaged in the chemiosmotic hypothesis.

The Role of Energy Coupling in the Transport of β-Galactosides by Escherichia coli

Abstract Lactose and o-nitrophenylgalactoside (NPG) were transported against considerable concentration gradients by ML 308-225, a mutant of Escherichia coli which lacks β-galactosidase but possesses a constitutive transport system for β-galactosides. The kinetics of influx of NPG during this accumulation were found to be the same as those found for this galactoside moving down a concentration gradient into ML 308, i.e. transport into the parent cell containing β-galactosidase. When cells of ML 308-225 were exposed to azide, azide plus iodoacetate, or dinitrophenol transport of these galactosides against a concentration gradient was completely abolished. However, the presence of functional membrane carriers in such inhibited cells was indicated by (a) very rapid equilibration of external and internal concentrations of galactoside, (b) inhibition of the rate of this equilibration by chemical analogues, and (c) transientaccumulation of substrate by washed cells preloaded with galactoside. All the evidence was consistent with the hypothesis that the same membrane carriers were involved in active transport by control cells and facilitated diffusion by poisoned cells. The most striking finding was that the addition of metabolic inhibitors reduced the Kt of exit about two orders of magnitude, whereas the Kt of entrance remained constant. It was inferred from these studies that energy coupling reduced the affinity of the carrier for its substrate on the inner surface of the plasma membrane. The possible physiological control of energy coupling of transport was discussed in the light of the present observations.