Removal of starch granule-associated proteins affects amyloglucosidase hydrolysis of rice starch granules.

Starch granule-associated proteins (SGAPs) include granule-surface proteins (SGSPs) and granule-channel proteins (SGCPs). To investigate impacts of SGAPs on amyloglucosidase (AMG) hydrolysis, waxy and non-waxy rice starches had their SGCPs or SGAPs removed. Removal of SGAPs or SGCPs did not affect morphology and amylopectin chain distribution but decreased relative crystallinity. Removal of SGAPs increased the digestion rate, AMG binding ability and pore diameter of hydrolyzed granules, and accelerated changes in relative crystallinity and destruction of crystalline region on hydrolysis. However, after removing SGCPs, AMG only bound to surface and attacked of the fingerprint of protein bodies on granules, with decreased hydrolysis rate. The degree of change in hydrolysis rate was not determined by SGCPs content of rice starch. These results implied that SGCPs had a more dominant role in AMG hydrolysis of rice starch than did SGSPs. This study provides novel information about the role of SGAPs in AMG hydrolysis mechanisms.

Characterization of the first planctomycetal outer membrane protein identifies a channel in the outer membrane of the anammox bacterium Kuenenia stuttgartiensis.

Planctomycetes are a bacterial phylum known for their complex intracellular compartmentalization. While most Planctomycetes have two compartments, the anaerobic ammonium oxidizing (anammox) bacteria contain three membrane-enclosed compartments. In contrast to a long-standing consensus, recent insights suggested the outermost Planctomycete membrane to be similar to a Gram-negative outer membrane (OM). One characteristic component that differentiates OMs from cytoplasmic membranes (CMs) is the presence of outer membrane proteins (OMPs) featuring a ß-barrel structure that facilitates passage of molecules through the OM. Although proteomic and genomic evidence suggested the presence of OMPs in several Planctomycetes, no experimental verification existed of the pore-forming function and localization of these proteins in the outermost membrane of these exceptional microorganisms. Here, we show via lipid bilayer assays that at least two typical OMP-like channel-forming proteins are present in membrane preparations of the anammox bacterium Kuenenia stuttgartiensis. One of these channel-forming proteins, the highly abundant putative OMP Kustd1878, was purified to homogeneity. Analysis of the channel characteristics via lipid bilayer assays showed that Kustd1878 forms a moderately cation-selective channel with a high current noise and an average single-channel conductance of about 170-190pS in 1M KCl. Antibodies were raised against the purified protein and immunogold localization indicated Kustd1878 to be present in the outermost membrane. Therefore, this work clearly demonstrates the presence of OMPs in anammox Planctomycetes and thus firmly adds to the emerging view that Planctomycetes have a Gram-negative cell envelope.

A binding-block ion selective mechanism revealed by a Na/K selective channel.

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na+/K+ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaIMet158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaIMet158 facilitate Na+/K+ pass through, which was defined as binding-block mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.

Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.

Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment.

A Low-Noise Transimpedance Amplifier for BLM-Based Ion Channel Recording.

High-throughput screening (HTS) using ion channel recording is a powerful drug discovery technique in pharmacology. Ion channel recording with planar bilayer lipid membranes (BLM) is scalable and has very high sensitivity. A HTS system based on BLM ion channel recording faces three main challenges: (i) design of scalable microfluidic devices; (ii) design of compact ultra-low-noise transimpedance amplifiers able to detect currents in the pA range with bandwidth >10 kHz; (iii) design of compact, robust and scalable systems that integrate these two elements. This paper presents a low-noise transimpedance amplifier with integrated A/D conversion realized in CMOS 0.35 µm technology. The CMOS amplifier acquires currents in the range ±200 pA and ±20 nA, with 100 kHz bandwidth while dissipating 41 mW. An integrated digital offset compensation loop balances any voltage offsets from Ag/AgCl electrodes. The measured open-input input-referred noise current is as low as 4 fA/√Hz at ±200 pA range. The current amplifier is embedded in an integrated platform, together with a microfluidic device, for current recording from ion channels. Gramicidin-A, α-haemolysin and KcsA potassium channels have been used to prove both the platform and the current-to-digital converter.

The role of lipids in mechanosensation.

The ability of proteins to sense membrane tension is pervasive in biology. A higher-resolution structure of the Escherichia coli small-conductance mechanosensitive channel MscS identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids, and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chains within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (with two acyl chains) from MscS pockets and trigger channel opening. We propose that the extent of acyl-chain interdigitation in these pockets determines the conformation of MscS. When interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable, and the channel gates.

Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin.

Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria.

CHEXVIS: a tool for molecular channel extraction and visualization.

BACKGROUND: Understanding channel structures that lead to active sites or traverse the molecule is important in the study of molecular functions such as ion, ligand, and small molecule transport. Efficient methods for extracting, storing, and analyzing protein channels are required to support such studies. Further, there is a need for an integrated framework that supports computation of the channels, interactive exploration of their structure, and detailed visual analysis of their properties. RESULTS: We describe a method for molecular channel extraction based on the alpha complex representation. The method computes geometrically feasible channels, stores both the volume occupied by the channel and its centerline in a unified representation, and reports significant channels. The representation also supports efficient computation of channel profiles that help understand channel properties. We describe methods for effective visualization of the channels and their profiles. These methods and the visual analysis framework are implemented in a software tool, CHEXVIS. We apply the method on a number of known channel containing proteins to extract pore features. Results from these experiments on several proteins show that CHEXVIS performance is comparable to, and in some cases, better than existing channel extraction techniques. Using several case studies, we demonstrate how CHEXVIS can be used to study channels, extract their properties and gain insights into molecular function. CONCLUSION: CHEXVIS supports the visual exploration of multiple channels together with their geometric and physico-chemical properties thereby enabling the understanding of the basic biology of transport through protein channels. The CHEXVIS web-server is freely available at http://vgl.serc.iisc.ernet.in/chexvis/ . The web-server is supported on all modern browsers with latest Java plug-in.

Limits and possibilities experienced by nurses in the treatment of women with chronic venous ulcers / Límites y posibilidades vividas por enfermeras en el tratamiento de mujeres con úlcera venosa crónica / Limites e possibilidades vivenciados por enfermeiras no tratamento de mulheres com úlcera venosa crônica

Objective To understand the experiences and expectations of nurses in the treatment of women with chronic venous ulcers. Method Phenomenological research was based on Alfred Schütz, whose statements were obtained in January, 2012, through semi-structured interviews with seven nurses. Results The nurse reveals the difficulties presented by the woman in performing self-care, the perceived limitations in the treatment anchored in motivation, and the values and beliefs of women. It showed professional frustration because venous leg ulcer recurrence, lack of inputs, interdisciplinary work and training of nursing staff. There was an expected adherence to the treatment of women, and it emphasized the need for ongoing care, supported self-care and standard practices in treatment. Conclusion That treatment of chronic venous leg ulcers constitutes a challenge that requires collective investment, involving women, professionals, managers and health institutions. .

Objetivo Comprender las experiencias y expectativas de enfermeras en el tratamiento de mujeres con úlcera venosa crónica. Método Investigación fenomenológica fundamentada en Alfred Schutz, que buscó Se realizó entrevista semiestructurada con siete enfermeras, en enero del 2012. Resultados La enfermera revela dificultades presentadas por la mujer para realizar el autocuidado, percibe limitaciones en el tratamiento relacionadas con la desmotivación, los valores y las creencias de las mujeres. Refiere frustración profesional debido a la recidiva de la lesión, a la falta de insumos, al deficiente trabajo interdisciplinar y a la limitada capacitación del equipo de enfermeras. Espera la adhesión de la mujer al tratamiento y resalta la necesidad del cuidado continuo, del autocuidado apoyado y de estandarizar conductas de tratamiento. Conclusión El tratamiento de la úlcera venosa crónica es un desafío que requiere contribución colectiva, involucrando a las mujeres, a los profesionales, a los gestores y a las instituciones de salud. .

Objetivo Compreender as experiências e expectativas de enfermeiras no tratamento de mulheres com úlcera venosa crônica na Atenção Primária à Saúde. Método Pesquisa fundamentada na fenomenologia social de Alfred Schütz, com depoimentos obtidos em janeiro de 2012, por meio de entrevista semiestruturada com sete enfermeiras. Resultados As enfermeiras revelam dificuldades apresentadas pelas mulheres com úlcera venosa crônica para realizar o autocuidado, percebem limitações na terapêutica ancoradas na desmotivação e nos valores e crenças das mulheres. Referem frustração profissional em razão da recidiva da lesão, falta de insumos e tecnologia, de trabalho interdisciplinar e da capacitação da equipe de enfermagem. Esperam a adesão das mulheres ao tratamento e ressaltam a necessidade do cuidado contínuo, do autocuidado apoiado e da padronização de condutas no tratamento. Conclusão O tratamento da úlcera venosa crônica constitui-se em um desafio que requer investimento coletivo, envolvendo a mulher, os profissionais, os gestores e as instituições de saúde. .

Use of Escherichia coli for the production and purification of membrane proteins.

Individual types of ion channels and other membrane proteins are typically expressed only at low levels in their native membranes, rendering their isolation by conventional purification techniques difficult. The heterologous over-expression of such proteins is therefore usually a prerequisite for their purification in amounts suitable for structural and for many functional investigations. The most straightforward expression host, suitable for prokaryote membrane proteins and some proteins from eukaryotes, is the bacterium Escherichia coli. Here we describe the use of this expression system for production of functionally active polytopic membrane proteins and methods for their purification by affinity chromatography in amounts up to tens of milligrams.

Isotope labeling strategies for analysis of an ion channel cytoplasmic domain by NMR spectroscopy.

As large, multimeric, integral membrane proteins, ion channels pose technical challenges to analysis by NMR spectroscopy. Here we present a strategy to overcome some of these technical hurdles, using a representative ion channel modulatory domain, the regulator of K(+) conductance (RCK) domain from a K(+) channel cloned from Thermoplasma volcanium. By introducing a mutation to limit the stoichiometry of the octameric RCK domain "gating ring" complex to its dimeric building block, NMR spectral resolution can be greatly improved. Here we present protocols for efficient production of highly deuterated, uniformly (15)N-labeled protein, as well as protein containing (15)N-labeling to specific amino acid types. These labeling strategies can be applied to improve spectral resolution and facilitate sequential resonance assignments.

Hydrophobic gating of mechanosensitive channel of large conductance evidenced by single-subunit resolution.

Mechanosensitive (MS) ion channels are membrane proteins that detect and respond to membrane tension in all branches of life. In bacteria, MS channels prevent cells from lysing upon sudden hypoosmotic shock by opening and releasing solutes and water. Despite the importance of MS channels and ongoing efforts to explain their functioning, the molecular mechanism of MS channel gating remains elusive and controversial. Here we report a method that allows single-subunit resolution for manipulating and monitoring "mechanosensitive channel of large conductance" from Escherichia coli. We gradually changed the hydrophobicity of the pore constriction in this homopentameric protein by modifying a critical pore residue one subunit at a time. Our experimental results suggest that both channel opening and closing are initiated by the transmembrane 1 helix of a single subunit and that the participation of each of the five identical subunits in the structural transitions between the closed and open states is asymmetrical. Such a minimal change in the pore environment seems ideal for a fast and energy-efficient response to changes in the membrane tension.

Bacterial mechanosensitive channels--MscS: evolution's solution to creating sensitivity in function.

The discovery of mechanosensing channels has changed our understanding of bacterial physiology. The mechanosensitive channel of small conductance (MscS) is perhaps the most intensively studied of these channels. MscS has at least two states: closed, which does not allow solutes to exit the cytoplasm, and open, which allows rapid efflux of solvent and solutes. The ability to appropriately open or close the channel (gating) is critical to bacterial survival. We briefly review the science that led to the isolation and identification of MscS. We concentrate on the structure-function relationship of the channel, in particular the structural and biochemical approaches to understanding channel gating. We highlight the troubling discrepancies between the various models developed to understand MscS gating.

Identification and characterization of a bacterial hydrosulphide ion channel.

The hydrosulphide ion (HS(-)) and its undissociated form, hydrogen sulphide (H(2)S), which are believed to have been critical to the origin of life on Earth, remain important in physiology and cellular signalling. As a major metabolite in anaerobic bacterial growth, hydrogen sulphide is a product of both assimilatory and dissimilatory sulphate reduction. These pathways can reduce various oxidized sulphur compounds including sulphate, sulphite and thiosulphate. The dissimilatory sulphate reduction pathway uses this molecule as the terminal electron acceptor for anaerobic respiration, in which process it produces excess amounts of H(2)S (ref. 4). The reduction of sulphite is a key intermediate step in all sulphate reduction pathways. In Clostridium and Salmonella, an inducible sulphite reductase is directly linked to the regeneration of NAD(+), which has been suggested to have a role in energy production and growth, as well as in the detoxification of sulphite. Above a certain concentration threshold, both H(2)S and HS(-) inhibit cell growth by binding the metal centres of enzymes and cytochrome oxidase, necessitating a release mechanism for the export of this toxic metabolite from the cell. Here we report the identification of a hydrosulphide ion channel in the pathogen Clostridium difficile through a combination of genetic, biochemical and functional approaches. The HS(-) channel is a member of the formate/nitrite transport family, in which about 50 hydrosulphide ion channels form a third subfamily alongside those for formate (FocA) and for nitrite (NirC). The hydrosulphide ion channel is permeable to formate and nitrite as well as to HS(-) ions. Such polyspecificity can be explained by the conserved ion selectivity filter observed in the channel's crystal structure. The channel has a low open probability and is tightly regulated, to avoid decoupling of the membrane proton gradient.

Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant.

Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.

Supramolecular complexes of the Agrobacterium tumefaciens virulence protein VirE2.

Virulence protein VirE2 from Agrobacterium tumefaciens is involved in plant infection by transferring a fragment of agrobacterial Ti plasmid ssT-DNA in complex with VirE2-VirD2 proteins into the plant cell nucleus. The VirE2 protein interactions with ssDNA and formation of VirE2 protein complexes in vitro and in silico have been studied. Using dynamic light scattering we found that purified recombinant protein VirE2 exists in buffer solution in the form of complexes of 2-4 protein molecules of 12-18 nm size. We used computer methods to design models of complexes consisting of two and four individual VirE2 proteins, and their dimensions were estimated. Dimensions of VirE2 complexes with ssDNA (550 and 700 nucleotide residues) were determined using transmission electron microscopy and dynamic light scattering. We found that in vitro, upon interaction with ssDNA recombinant protein, VirE2 is able to alter conformation of the latter by shortening the initial length of the ssDNA.

Identification and characterization of uncoupling protein 4 in fat body and muscle mitochondria from the cockroach Gromphadorhina cocquereliana.

We have identified and characterized an uncoupling protein in mitochondria isolated from leg muscle and from fat body, an insect analogue tissue of mammalian liver and adipose tissue, of the cockroach Gromphadorhina coquereliana (GcUCP). This is the first functional characterization of UCP activity in isolated insect mitochondria. Bioenergetic studies clearly indicate UCP function in both insect tissues. In resting (non-phosphorylating) mitochondria, cockroach GcUCP activity was stimulated by the addition of micromolar concentrations of palmitic acid and inhibited by the purine nucleotide GTP. Moreover, in phosphorylating mitochondria, GcUCP activity was able to divert energy from oxidative phosphorylation. Functional studies indicate a higher activity of GcUCP-mediated uncoupling in cockroach muscle mitochondria compared to fat body mitochondria. GcUCP activation by palmitic acid resulted in a decrease in superoxide anion production, suggesting that protection against mitochondrial oxidative stress may be a physiological role of UCPs in insects. GcUCP protein was immunodetected using antibodies raised against human UCP4 as a single band of around 36 kDa. GcUCP protein expression in cockroach muscle mitochondria was significantly higher compared to mitochondria isolated from fat body. LC-MS/MS analyses revealed 100% sequence identities for peptides obtained from GcUCP to UCP4 isoforms from D. melanogaster (the highest homology), human, rat or other insect mitochondria. Therefore, it can be proposed that cockroach GcUCP corresponds to the UCP4 isoforms of other animals.

pH-dependent gating in a FocA formate channel.

The formate transporter FocA was described to switch its mode of operation from a passive export channel at high external pH to a secondary active formate/H(+) importer at low pH. The crystal structure of Salmonella typhimurium FocA at pH 4.0 shows that this switch involves a major rearrangement of the amino termini of individual protomers in the pentameric channel. The amino-terminal helices open or block transport in a concerted, cooperative action that indicates how FocA is gated in a pH-dependent way. Electrophysiological studies show that the protein acts as a specific formate channel at pH 7.0 and that it closes upon a shift of pH to 5.1.

Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels.

BACKGROUND: Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies. PRINCIPAL FINDINGS: We describe the homologous and heterologous expression of 10 different targets from the three major MP classes--G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2-0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter--both involved in synaptic transmission--showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies. SIGNIFICANCE: We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies.