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1.
Rapid Commun Mass Spectrom ; 32(24): 2129-2140, 2018 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-30252972

RESUMO

RATIONALE: Microbial growth rate is an important physiological parameter that is challenging to measure in situ, partly because microbes grow slowly in many environments. Recently, it has been demonstrated that generation times of S. aureus in cystic fibrosis (CF) infections can be determined by D2 O-labeling of actively synthesized fatty acids. To improve species specificity and allow growth rate monitoring for a greater range of pathogens during the treatment of infections, it is desirable to accurately quantify trace incorporation of deuterium into phospholipids. METHODS: Lipid extracts of D2 O-treated E. coli cultures were measured on liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) instruments equipped with time-of-flight (TOF) and orbitrap mass analyzers, and used for comparison with the analysis of fatty acids by isotope-ratio gas chromatography (GC)/MS. We then developed an approach to enable tracking of lipid labeling, by following the transition from stationary into exponential growth in pure cultures. Lastly, we applied D2 O-labeling lipidomics to clinical samples from CF patients with chronic lung infections. RESULTS: Lipidomics facilitates deuterium quantification in lipids at levels that are useful for many labeling applications (>0.03 at% D). In the E. coli cultures, labeling dynamics of phospholipids depend largely on their acyl chains and between phospholipids we notice differences that are not obvious from absolute concentrations alone. For example, cyclopropyl-containing lipids reflect the regulation of cyclopropane fatty acid synthase, which is predominantly expressed at the beginning of stationary phase. The deuterium incorporation into a lipid that is specific for S. aureus in CF sputum indicates an average generation time of the pathogen on the order of one cell doubling per day. CONCLUSIONS: This study demonstrates how trace level measurement of stable isotopes in intact lipids can be used to quantify lipid metabolism in pure cultures and provides guidelines that enable growth rate measurements in microbiome samples after incubation with a low percentage of D2 O.


Assuntos
Fibrose Cística/microbiologia , Deutério/química , Escherichia coli/crescimento & desenvolvimento , Ácidos Graxos/química , Staphylococcus aureus/crescimento & desenvolvimento , Água/química , Cromatografia Líquida , Deutério/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Humanos , Cinética , Metabolismo dos Lipídeos , Espectrometria de Massas por Ionização por Electrospray , Escarro/química , Escarro/microbiologia , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Água/metabolismo
2.
Biochemistry ; 56(32): 4219-4234, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28656748

RESUMO

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.


Assuntos
Monofosfato de Adenosina/química , Antiportadores de Potássio-Hidrogênio/química , Dobramento de Proteína , Multimerização Proteica , Shewanella/química , Monofosfato de Adenosina/genética , Monofosfato de Adenosina/metabolismo , Antiportadores de Potássio-Hidrogênio/genética , Antiportadores de Potássio-Hidrogênio/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Estrutura Quaternária de Proteína , Shewanella/genética , Shewanella/metabolismo
3.
Biochemistry ; 54(29): 4519-30, 2015 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-26126964

RESUMO

Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons. In the MscS crystal structure (Protein Data Bank entry 2oau ), a narrow, hydrophobic opening is visible in the crystal structure, and it has been proposed that a vapor lock created by the hydrophobic seals, L105 and L109, is the barrier to water and ions. Tryptophan scanning mutagenesis has proven to be a highly valuable tool for the analysis of channel structure. Here Trp residues were introduced along the pore-forming TM3a helix and in selected other parts of the protein. Mutants were investigated for their expression, stability, and activity and as fluorescent probes of the physical properties along the length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state fluorescence anisotropy data are consistent with significant homo-Förster resonance energy transfer between tryptophan residues from different subunits within the narrow pore. The data provide new insights into MscS structure and gating.


Assuntos
Proteínas de Escherichia coli/genética , Canais Iônicos/genética , Proteínas de Escherichia coli/química , Polarização de Fluorescência , Canais Iônicos/química , Lipossomos/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Permeabilidade , Fosfatidilcolinas/química , Conformação Proteica , Estabilidade Proteica , Triptofano/genética
4.
Proc Natl Acad Sci U S A ; 109(40): E2675-82, 2012 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-23012406

RESUMO

The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key secondary structural elements specifically chosen to discriminate between the competing models. The resulting pulsed electron-electron double resonance (PELDOR) spectra matched predicted distance distributions for the open crystal structure of MscS. The fit for the predictions by structural models of MscS derived by other techniques was not convincing. The assignment of MscS as open in detergent by PELDOR was unexpected but is supported by two crystal structures of spin-labeled MscS. PELDOR is therefore shown to be a powerful experimental tool to interrogate the conformation of transmembrane regions of integral membrane proteins.


Assuntos
Proteínas de Escherichia coli/química , Canais Iônicos/química , Modelos Moleculares , Conformação Proteica , Análise Espectral/métodos , Western Blotting , Cromatografia em Gel , Cristalografia , Espectroscopia de Ressonância de Spin Eletrônica , Mutagênese , Técnicas de Patch-Clamp , Análise de Sequência de DNA , Marcadores de Spin
5.
Biophys J ; 106(4): 834-42, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24559986

RESUMO

Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void. The relevance of this void to the functional form of MscS in the bilayer is the subject of debate. Here, we report PELDOR measurements of MscS reconstituted into two lipid bilayer systems: nanodiscs and bicelles. The distance measurements from multiple mutants derived from the PELDOR data are consistent with the detergent-solution arrangement of the protein. We conclude, therefore, that the relative positioning of the transmembrane helices is preserved in mimics of the cell bilayer, and that the apparent voids are not an artifact of detergent solution but a property of the protein that will have to be accounted for in any molecular mechanism of gating.


Assuntos
Proteínas de Escherichia coli/química , Canais Iônicos/química , Bicamadas Lipídicas/metabolismo , Sequência de Aminoácidos , Cristalografia , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Bicamadas Lipídicas/química , Dados de Sequência Molecular , Estrutura Terciária de Proteína
6.
Biochemistry ; 53(12): 1982-92, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24601535

RESUMO

The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of ∼10-20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10-20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system.


Assuntos
Proteínas de Escherichia coli/química , Glutationa/análogos & derivados , Antiportadores de Potássio-Hidrogênio/química , Potássio/química , Succinimidas/química , Transporte Biológico Ativo/fisiologia , Proteínas de Escherichia coli/metabolismo , Glutationa/química , Glutationa/metabolismo , Ativação do Canal Iônico/fisiologia , Ligantes , Potássio/metabolismo , Antiportadores de Potássio-Hidrogênio/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Shewanella/química , Shewanella/metabolismo , Succinimidas/metabolismo
7.
Mol Microbiol ; 88(5): 936-50, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23646895

RESUMO

Methylglyoxal (MG) elicits activation of K(+) efflux systems to protect cells against the toxicity of the electrophile. ChIP-chip targeting RNA polymerase, supported by a range of other biochemical measurements and mutant creation, was used to identify genes transcribed in response to MG and which complement this rapid response. The SOS DNA repair regulon is induced at cytotoxic levels of MG, even when exposure to MG is transient. Glyoxalase I alone among the core MG protective systems is induced in response to MG exposure. Increased expression is an indirect consequence of induction of the upstream nemRA operon, encoding an enzyme system that itself does not contribute to MG detoxification. Moreover, this induction, via nemRA only occurs when cells are exposed to growth inhibitory concentrations of MG. We show that the kdpFABCDE genes are induced and that this expression occurs as a result of depletion of cytoplasmic K(+) consequent upon activation of the KefGB K(+) efflux system. Finally, our analysis suggests that the transcriptional changes in response to MG are a culmination of the damage to DNA and proteins, but that some integrate specific functions, such as DNA repair, to augment the allosteric activation of the main protective system, KefGB.


Assuntos
Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Lactoilglutationa Liase/biossíntese , Óperon , Aldeído Pirúvico/toxicidade , Estresse Fisiológico , Transcrição Gênica , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas de Escherichia coli/genética , Genes Bacterianos/genética , Lactoilglutationa Liase/genética , Resposta SOS em Genética , Fatores de Transcrição/genética
8.
Proc Natl Acad Sci U S A ; 107(28): 12664-9, 2010 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-20616037

RESUMO

We describe a mechanosensitive (MS) channel that has mechanosensitive channel of miniconductance (MscM) activity, and displays unique properties with respect to gating. Mechanosensitive channels respond to membrane tension, are ubiquitous from bacteria to man, and exhibit a great diversity in structure and function. These channels protect Bacteria and Archaea against hypoosmotic shock and are critical determinants of shape in chloroplasts. Given the dominant roles played in bacteria by the mechanosensitive channel of small conductance (MscS) and the mechanosensitive channel of large conductance (MscL), the role of the multiple MS channel homologs observed in most organisms remains obscure. Here we demonstrate that a MscS homolog, YbdG, extends the range of hypoosmotic shock that Escherichia coli cells can survive, but its expression level is insufficient to protect against severe shocks. Overexpression of the YbdG protein provides complete protection. Transcription and translation of the ybdG gene are enhanced by osmotic stress consistent with a role for the protein in survival of hypoosmotic shock. Measurement of the conductance of the native channel by standard patch clamp methods was not possible. However, a fully functional YbdG mutant channel, V229A, exhibits a conductance in membrane patches consistent with MscM activity. We find that MscM activities arise from more than one gene product because ybdG deletion mutants still exhibit an occasional MscM-like conductance. We propose that ybdG encodes a low-abundance MscM-type MS channel, which in cells relieves low levels of membrane tension, obviating the need to activate the major MS channels, MscS and MscL.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Archaea/genética , Archaea/metabolismo , Deleção de Sequência
9.
Proc Natl Acad Sci U S A ; 107(46): 19784-9, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21041667

RESUMO

Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. We find that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. We demonstrate a major structural change on the binding of an activating ligand to a KTN-domain protein. Analysis of the regulatory interactions suggests strategies to disrupt pathogen potassium and pH homeostasis.


Assuntos
Escherichia coli/metabolismo , Ativação do Canal Iônico/fisiologia , Potássio/metabolismo , Sequência de Aminoácidos , Transporte Biológico/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glutationa/análogos & derivados , Glutationa/metabolismo , Glutationa/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Succinimidas/farmacologia
10.
J Bacteriol ; 194(18): 4802-9, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22685280

RESUMO

Single-celled organisms must survive exposure to environmental extremes. Perhaps one of the most variable and potentially life-threatening changes that can occur is that of a rapid and acute decrease in external osmolarity. This easily translates into several atmospheres of additional pressure that can build up within the cell. Without a protective mechanism against such pressures, the cell will lyse. Hence, most microbes appear to possess members of one or both families of bacterial mechanosensitive channels, MscS and MscL, which can act as biological emergency release valves that allow cytoplasmic solutes to be jettisoned rapidly from the cell. While this is undoubtedly a function of these proteins, the discovery of the presence of MscS homologues in plant organelles and MscL in fungus and mycoplasma genomes may complicate this simplistic interpretation of the physiology underlying these proteins. Here we compare and contrast these two mechanosensitive channel families, discuss their potential physiological roles, and review some of the most relevant data that underlie the current models for their structure and function.


Assuntos
Fenômenos Fisiológicos Bacterianos , Canais Iônicos/metabolismo , Mecanotransdução Celular , Pressão Osmótica , Estresse Fisiológico , Canais Iônicos/química , Modelos Moleculares
11.
Biophys J ; 100(4): 814-21, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21320424

RESUMO

DNA damage is a hazard all cells must face, and evolution has created a number of mechanisms to repair damaged bases in the chromosome. Paradoxically, many of these repair mechanisms can create double-strand breaks in the DNA molecule which are fatal to the cell. This indicates that the connection between DNA repair and death is far from straightforward, and suggests that the repair mechanisms can be a double-edged sword. In this report, we formulate a mathematical model of the dynamics of DNA damage and repair, and we obtain analytical expressions for the death rate. We predict a counterintuitive relationship between survival and repair. We can discriminate between two phases: below a critical threshold in the number of repair enzymes, the half-life decreases with the number of repair enzymes, but becomes independent of the number of repair enzymes above the threshold. We are able to predict quantitatively the dependence of the death rate on the damage rate and other relevant parameters. We verify our analytical results by simulating the stochastic dynamics of DNA damage and repair. Finally, we also perform an experiment with Escherichia coli cells to test one of the predictions of our model.


Assuntos
Dano ao DNA , Reparo do DNA , Escherichia coli/citologia , Escherichia coli/metabolismo , Viabilidade Microbiana , Modelos Biológicos , Simulação por Computador , Reparo do DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Aldeído Pirúvico/toxicidade , Processos Estocásticos
12.
J Bacteriol ; 193(18): 4925-32, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21742892

RESUMO

Escherichia coli and many other Gram-negative pathogenic bacteria protect themselves from the toxic effects of electrophilic compounds by using a potassium efflux system (Kef). Potassium efflux is coupled to the influx of protons, which lowers the internal pH and results in immediate protection. The activity of the Kef system is subject to complex regulation by glutathione and its S conjugates. Full activation of KefC requires a soluble ancillary protein, KefF. This protein has structural similarities to oxidoreductases, including human quinone reductases 1 and 2. Here, we show that KefF has enzymatic activity as an oxidoreductase, in addition to its role as the KefC activator. It accepts NADH and NADPH as electron donors and quinones and ferricyanide (in addition to other compounds) as acceptors. However, typical electrophilic activators of the Kef system, e.g., N-ethyl maleimide, are not substrates. If the enzymatic activity is disrupted by site-directed mutagenesis while retaining structural integrity, KefF is still able to activate the Kef system, showing that the role as an activator is independent of the enzyme activity. Potassium efflux assays show that electrophilic quinones are able to activate the Kef system by forming S conjugates with glutathione. Therefore, it appears that the enzymatic activity of KefF diminishes the redox toxicity of quinones, in parallel with the protection afforded by activation of the Kef system.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Oxirredutases/metabolismo , Benzoquinonas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Ferricianetos/metabolismo , Humanos , Mutagênese Sítio-Dirigida , NAD/metabolismo , NAD(P)H Desidrogenase (Quinona)/genética , NADP/metabolismo , Oxirredutases/genética , Potássio/metabolismo , Canais de Potássio/metabolismo , Subunidades Proteicas/metabolismo
13.
J Biol Chem ; 285(8): 5377-84, 2010 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-20037156

RESUMO

Structural changes in channel proteins give critical insights required for understanding the gating transitions that underpin function. Tryptophan (Trp) is uniquely sensitive to its environment and can be used as a reporter of conformational changes. Here, we have used site-directed Trp insertion within the pore helices of the small mechanosensitive channel protein, MscS, to monitor conformational transitions. We show that Trp can be inserted in place of Leu at the two pore seal positions, Leu(105) and Leu(109), resulting in functional channels. Using Trp(105) as a probe, we demonstrate that the A106V mutation causes a modified conformation in the purified channel protein consistent with a more open state in solution. Moreover, we show that solubilized MscS changes to a more open conformation in the presence of phospholipids or their lysoforms.


Assuntos
Proteínas de Escherichia coli/química , Canais Iônicos/química , Triptofano/química , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ativação do Canal Iônico/fisiologia , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mutação de Sentido Incorreto , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Estrutura Secundária de Proteína/fisiologia , Espectrometria de Fluorescência , Triptofano/genética , Triptofano/metabolismo
14.
Mol Microbiol ; 78(6): 1577-90, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21143325

RESUMO

Survival of exposure to methylglyoxal (MG) in Gram-negative pathogens is largely dependent upon the operation of the glutathione-dependent glyoxalase system, consisting of two enzymes, GlxI (gloA) and GlxII (gloB). In addition, the activation of the KefGB potassium efflux system is maintained closed by glutathione (GSH) and is activated by S-lactoylGSH (SLG), the intermediate formed by GlxI and destroyed by GlxII. Escherichia coli mutants lacking GlxI are known to be extremely sensitive to MG. In this study we demonstrate that a ΔgloB mutant is as tolerant of MG as the parent, despite having the same degree of inhibition of MG detoxification as a ΔgloA strain. Increased expression of GlxII from a multicopy plasmid sensitizes E. coli to MG. Measurement of SLG pools, KefGB activity and cytoplasmic pH shows these parameters to be linked and to be very sensitive to changes in the activity of GlxI and GlxII. The SLG pool determines the activity of KefGB and the degree of acidification of the cytoplasm, which is a major determinant of the sensitivity to electrophiles. The data are discussed in terms of how cell fate is determined by the relative abundance of the enzymes and KefGB.


Assuntos
Escherichia coli/metabolismo , Glutationa/análogos & derivados , Lactoilglutationa Liase/metabolismo , Aldeído Pirúvico/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glutationa/metabolismo , Lactoilglutationa Liase/genética , Viabilidade Microbiana , Antiportadores de Potássio-Hidrogênio/genética , Antiportadores de Potássio-Hidrogênio/metabolismo , Aldeído Pirúvico/farmacologia
15.
Biochem Soc Trans ; 39(3): 733-40, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21599642

RESUMO

Mechanosensitive channels sense and respond to changes in bilayer tension. In many respects, this is a unique property: the changes in membrane tension gate the channel, leading to the transient formation of open non-selective pores. Pore diameter is also high for the bacterial channels studied, MscS and MscL. Consequently, in cells, gating has severe consequences for energetics and homoeostasis, since membrane depolarization and modification of cytoplasmic ionic composition is an immediate consequence. Protection against disruption of cellular integrity, which is the function of the major channels, provides a strong evolutionary rationale for possession of such disruptive channels. The elegant crystal structures for these channels has opened the way to detailed investigations that combine molecular genetics with electrophysiology and studies of cellular behaviour. In the present article, the focus is primarily on the structure of MscS, the small mechanosensitive channel. The description of the structure is accompanied by discussion of the major sites of channel-lipid interaction and reasoned, but limited, speculation on the potential mechanisms of tension sensing leading to gating.


Assuntos
Bactérias/metabolismo , Ativação do Canal Iônico/fisiologia , Bicamadas Lipídicas/química , Mecanotransdução Celular/fisiologia , Bactérias/citologia , Escherichia coli/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , Estresse Mecânico
16.
Nat Struct Mol Biol ; 12(2): 113-9, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15665866

RESUMO

The crystal structure of an open form of the Escherichia coli MscS mechanosensitive channel was recently solved. However, the conformation of the closed state and the gating transition remain uncharacterized. The pore-lining transmembrane helix contains a conserved glycine- and alanine-rich motif that forms a helix-helix interface. We show that introducing 'knobs' on the smooth glycine face by replacing glycine with alanine, and substituting conserved alanines with larger residues, increases the pressure required for gating. Creation of a glycine-glycine interface lowers activation pressure. The importance of residues Gly104, Ala106 and Gly108, which flank the hydrophobic seal, is demonstrated. A new structural model is proposed for the closed-to-open transition that involves rotation and tilt of the pore-lining helices. Introduction of glycine at Ala106 validated this model by acting as a powerful suppressor of defects seen with mutations at Gly104 and Gly108.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Glicina/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Alanina/genética , Alanina/metabolismo , Eletrofisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Glicina/genética , Canais Iônicos/genética , Modelos Moleculares , Mutação/genética , Fenótipo , Estrutura Terciária de Proteína
17.
Biophys J ; 94(8): 3003-13, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18065458

RESUMO

Mechanosensitive channels rescue bacterial cells from a fate of lysis when they transfer from a high- to low-osmolarity environment. Of three Escherichia coli mechanosensitive proteins studied to date, only MscS-Ec demonstrates a small anionic preference and a desensitized, nonconducting state under sustained pressure. Little is known about the mechanisms generating these distinctive properties. Eliminating the sole positive charge in the MscS-Ec pore region (Arg(88)) did not alter anionic preference. Adding positive charges at either end of the pore did not augment anionic preference, and placing negative charges within the pore did not diminish it. Thus, pore charges do not control this characteristic. However, from this analysis we identified mutations in the hinge region of the MscS-Ec pore helix (at Gly(113)) that profoundly affected ability of the channel to desensitize. Substitution with nonpolar (Ala, Pro) or polar (Asp, Arg, Ser) residues inhibited transition to the desensitized state. Interestingly, Gly(113) replaced with Met did not impede desensitization. Thus, although Gly is not specifically required at position 113, MscS desensitization is strongly influenced by the residue situated here. Mutations at residues further into the pore also regulated desensitization. Transition to this unique mechanosensitive channel state is discussed in terms of existing data.


Assuntos
Membrana Celular/fisiologia , Proteínas de Escherichia coli/genética , Ativação do Canal Iônico/fisiologia , Canais Iônicos/genética , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Mutagênese Sítio-Dirigida , Substituição de Aminoácidos , Membrana Celular/química , Simulação por Computador , Proteínas de Escherichia coli/química , Canais Iônicos/química , Bicamadas Lipídicas/química , Modelos Químicos , Modelos Moleculares , Porosidade , Estresse Mecânico
18.
Microbiol Mol Biol Rev ; 67(1): 66-85, table of contents, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12626684

RESUMO

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Mecanotransdução Celular , Filogenia , Sequência de Aminoácidos , Sequência Conservada , Proteínas de Escherichia coli/genética , Canais Iônicos/genética , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica
19.
Methods Mol Biol ; 424: 167-85, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18369862

RESUMO

High-resolution 2-dimensional gel electrophoresis (2DGE) is a key technology in the analysis of cellular proteomes particularly in the field of microbiology. However, the restricted resolution of 2DGE and the limited dynamic range of established staining methods limit its usefulness for characterising low abundance proteins. Consequently, methods have been developed to either enrich for low abundance proteins directly or to deplete the highly abundant proteins present in complex samples. We present a protocol for affinity chromatography on reactive dye resins for the analysis of the Escherichia coli proteome. Using a range of commercially available reactive dye resins in a traditional chromatography system we were able to enrich low abundance proteins to levels suitable for their reliable detection and, most importantly, their identification using standard peptide mass mapping and MALDI-TOF MS methods. Under the chromatography conditions employed up to 4.42% of the proteins present in the total nonfractionated E. coli cell lysates bound to the reactive dye column and were subsequently eluted by 1.5 M NaCl. Of the bound proteins approximately 50% were considered to be enriched compared to the nonfractionated cell lysate. The ability to detect low abundance proteins was due to a combination of the specific enrichment of the proteins themselves as well as the depletion of highly abundant cellular proteins, which otherwise obscured the low abundance proteins. There was evidence of some selectivity between the different reactive dye resins for particular proteins. However, the selection of suitable dye resins to selectively enrich for particular classes of proteins remains largely empirical at this time.


Assuntos
Cromatografia Líquida/métodos , Corantes/química , Proteínas de Escherichia coli/isolamento & purificação , Escherichia coli/química , Proteoma/isolamento & purificação , Escherichia coli/metabolismo , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/química , Proteoma/análise , Proteoma/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
20.
Methods Enzymol ; 428: 47-61, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17875411

RESUMO

Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypoosmotic shock. Bacteria that have been diluted from high osmolarity medium into dilute solution are required to cope with sudden water influx associated with an osmotic imbalance equivalent to 10 to 14 atm. The cell wall is only poorly expansive and the cytoplasmic membrane even less so. Thus, swelling is not an option and the cell must rapidly eject solutes to diminish the osmotic gradient and thereby preserve structural integrity. This chapter describes cellular assays of MS channel function and their interpretation.


Assuntos
Fenômenos Fisiológicos Bacterianos , Canais Iônicos/fisiologia , Mecanotransdução Celular/fisiologia , Ativação do Canal Iônico , Canais Iônicos/genética , Pressão Osmótica
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