Identification of Subunits a, b, andc 1 from Acetobacterium woodiiNa+-F1F0-ATPase

Aufurth, Sascha; Müller, Volker

doi:10.1074/jbc.m005134200

Cited by 36 publications

(8 citation statements)

References 33 publications

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“…It can therefore be assumed that also in this case the complexity was too high and a prefractionation of complex protein mixtures is recommended before BNE. Similar results showing only a few protein bands in a smeary background were obtained when total membrane proteins of Agrobacterium species were separated by BNE [33,35,36]. Another deviation and possible explanation for the failure was that membrane solubilizate was used after detergent exchange.…”

Section: Bne and Native Electrophoresissupporting

confidence: 78%

Agarose isoelectric focusing can improve resolution of membrane proteins in the two‐dimensional electrophoresis of bacterial proteins

2006

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2-D separation of bacterial membrane proteins is still difficult despite using high-resolution IPG-IEF/SDS-PAGE. We were searching for alternative methods to avoid typical problems such as precipitation, low solubility, and aggregation of membrane proteins in the 1-D separation with IPG-IEF. Blue native electrophoresis (BNE) and agarose IEF (A-IEF) were tested for their separation capacity and their capability of replacing IPG-IEF in the first dimension. SDS-PAGE was chosen for the second dimension on account of its outstanding resolution. We could confirm that only A-IEF was a useful replacement for the IPG-IEF in the first dimension resulting in 2-D protein distributions with additional membrane protein spots not being found after IPG-IEF/SDS-PAGE. A second interesting result was that the agarose IEF mediates the possibility of separation of membrane proteins in a partially native state in the first dimension. This native A-IEF resulted in drastically changed spot patterns with an acidic shift of nearly all spots and divergent distribution of proteins compared to non-native A-IEF and IPG-IEF. We found out that native and non-native A-IEF are powerful tools to supplement IPG-IEF/SDS-PAGE.

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Section: Bne and Native Electrophoresissupporting

confidence: 78%

Agarose isoelectric focusing can improve resolution of membrane proteins in the two‐dimensional electrophoresis of bacterial proteins

2006

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“…These are also arranged as a ring‐shaped oligomer, composed of several (about 10–14; Ferguson, 2000) identical, small hydrophobic proteins (‘proteolipids’). The stability of the F 0 part of several ATPases in the presence of strong detergents has been reported to be extremely high (Laubinger and Dimroth, 1988; Neumann et al ., 1998; Aufurth et al ., 2000; Lingl et al ., 2003), very much like the stability of the oligomers of the I. hospitalis outer membrane protein Imp1227, observed in this study. In both cases, denaturation at temperatures at or below 100°C was not sufficient to yield a protein band with a mobility in SDS‐PAGE as expected for the monomeric protein.…”

Section: Discussionmentioning

confidence: 99%

The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore‐forming complex

Burghardt

Näther

Junglas

et al. 2006

Molecular Microbiology

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SummaryThe membrane protein Imp1227 (Ignicoccus outer membrane protein; Imp1227) is the main protein constituent of the unique outer sheath of the hyperthermophilic, chemolithoautotrophic Archaeum Ignicoccus hospitalis. This outer sheath is the so far only known example for an asymmetric bilayer among the Archaea and is named 'outer membrane'. With its molecular mass of only 6.23 kDa, Imp1227 is found to be incorporated into the outer membrane in form of large, stable complexes. When separated by SDS-PAGE, they exhibit apparent masses of about 150, 50, 45 and 35 kDa. Dissociation into the monomeric form is achieved by treatment with SDS-containing solutions at temperatures at or above 113°C. Electron micrographs of negatively stained samples confirm that isolated membranes are tightly packed with round complexes, about 7 nm in diameter, with a central, stain-filled 2 nm pore; a local two-dimensional crystalline arrangement in form of small patches can be detected by tomographic reconstruction. The comparison of the nucleotide and amino acid sequence of Imp1227 with public databases showed no reliable similarities with known proteins. Using secondary structure prediction and molecular modelling, an a-helical transmembrane domain is proposed; for the oligomer, a ring-shaped nonamer with a central 2 nm pore is a likely arrangement.

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“…Furthermore, the DDM-solubilized respiratory complex I of E. coli [376,377] as well as various protein complexes of Agrobacterium strains solubilized with DDM [378,379] or digitonin [380] were characterised after BN-PAGE separation. The subunit composition of the Na 1 -F O F 1 -ATP Synthase (,590 kDa) of Acetobacterium woodii was determined with 2-D BN/SDS-PAGE after solubilization with various detergents leading to consistent results [381].…”

Section: Protein Complexes In Prokaryotesmentioning

confidence: 99%

Detection and analysis of protein–protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes

2006

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It is an essential and challenging task to unravel protein-protein interactions in their actual in vivo context. Native gel systems provide a separation platform allowing the analysis of protein complexes on a rather proteome-wide scale in a single experiment. This review focus on blue-native (BN)-PAGE as the most versatile and successful gel-based approach to separate soluble and membrane protein complexes of intricate protein mixtures derived from all biological sources. BN-PAGE is a charge-shift method with a running pH of 7.5 relying on the gentle binding of anionic CBB dye to all membrane and many soluble protein complexes, leading to separation of protein species essentially according to their size and superior resolution than other fractionation techniques can offer. The closely related colorless-native (CN)-PAGE, whose applicability is restricted to protein species with intrinsic negative net charge, proved to provide an especially mild separation capable of preserving weak protein-protein interactions better than BN-PAGE. The essential conditions determining the success of detecting protein-protein interactions are the sample preparations, e.g. the efficiency/mildness of the detergent solubilization of membrane protein complexes. A broad overview about the achievements of BN- and CN-PAGE studies to elucidate protein-protein interactions in organelles and prokaryotes is presented, e.g. the mitochondrial protein import machinery and oxidative phosphorylation supercomplexes. In many cases, solubilization with digitonin was demonstrated to facilitate an efficient and particularly gentle extraction of membrane protein complexes prone to dissociation by treatment with other detergents. In general, analyses of protein interactomes should be carried out by both BN- and CN-PAGE.

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Identification of Subunits a, b, andc 1 from Acetobacterium woodiiNa+-F1F0-ATPase

Cited by 36 publications

References 33 publications

Agarose isoelectric focusing can improve resolution of membrane proteins in the two‐dimensional electrophoresis of bacterial proteins

Agarose isoelectric focusing can improve resolution of membrane proteins in the two‐dimensional electrophoresis of bacterial proteins

The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore‐forming complex

Detection and analysis of protein–protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes

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