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Native Gel Electrophoresis (native + gel_electrophoresis)
Selected AbstractsErrata: Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexesELECTROPHORESIS, Issue 24 2008Frank Krause Dr. No abstract is available for this article. [source] Identification and characterization of cytochrome bc1 subcomplexes in mitochondria from yeast with single and double deletions of genes encoding cytochrome bc1 subunitsFEBS JOURNAL, Issue 17 2007Vincenzo Zara We have examined the status of the cytochrome bc1 complex in mitochondrial membranes from yeast mutants in which genes for one or more of the cytochrome bc1 complex subunits were deleted. When membranes from wild-type yeast were resolved by native gel electrophoresis and analyzed by immunodecoration, the cytochrome bc1 complex was detected as a mixed population of enzymes, consisting of cytochrome bc1 dimers, and ternary complexes of cytochrome bc1 dimers associated with one and two copies of the cytochrome c oxidase complex. When membranes from the deletion mutants were resolved and analyzed, the cytochrome bc1 dimer was not associated with the cytochrome c oxidase complex in many of the mutant membranes, and membranes from some of the mutants contained a common set of cytochrome bc1 subcomplexes. When these subcomplexes were fractionated by SDS/PAGE and analyzed with subunit-specific antibodies, it was possible to recognize a subcomplex consisting of cytochrome b, subunit 7 and subunit 8 that is apparently associated with cytochrome c oxidase early in the assembly process, prior to acquisition of the remaining cytochrome bc1 subunits. It was also possible to identify a subcomplex consisting of subunit 9 and the Rieske protein, and two subcomplexes containing cytochrome c1 associated with core protein 1 and core protein 2, respectively. The analysis of all the cytochrome bc1 subcomplexes with monospecific antibodies directed against Bcs1p revealed that this chaperone protein is involved in a late stage of cytochrome bc1 complex assembly. [source] Isolation and characterization of a Lactobacillus amylovorus mutant depleted in conjugated bile salt hydrolase activity: relation between activity and bile salt resistanceJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2000J.P. Grill Growth experiments were conducted on Lactobacillus amylovorus DN-112 053 in batch culture, with or without pH regulation. Conjugated bile salt hydrolase (CBSH) activity was examined as a function of culture growth. The CBSH activity increased during growth but its course depended on bile salts type and culture conditions. A Lact. amylovorus mutant was isolated from the wild-type strain of Lact. amylovorus DN-112 053 after mutagenesis with N-methyl-N,-nitro-N-nitrosoguanidine. An agar plate assay was used to detect mutants without CBSH activity. In resting cell experiments, the strain showed reduced activity. Differences between growth parameters determined for wild-type and mutant strains were not detected. Comparative native gel electrophoresis followed by CBSH activity staining demonstrated the loss of proteins harbouring this activity in the mutant. Four protein bands corresponding to CBSH were observed in the wild-type strain but only one was detected in the mutant. The specific growth rate of the mutant strain was affected more by bile salts than the wild-type strain. Nevertheless, bile was more toxic for the wild-type strain. In viability studies in the presence of nutrients, it was demonstrated that glycodeoxycholic acid exerted a higher toxicity than taurodeoxycholic acid in a pH-dependent manner. No difference was apparent between the two strains. In the absence of nutrients, the wild-type strain died after 2 h whereas no effect was observed for the mutant. The de-energization experiments performed using the ionophores nigericin and valinomycin suggested that the chemical potential of protons (Z,pH) was involved in Lactobacillus bile salt resistance. [source] Detection and structural features of the ,B2-B3-crystallin heterodimer by radical probe mass spectrometry (RP-MS)JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2009Hélène Diemer Abstract The predilection of the ,-crystallin B2 subunit to interact with the ,B3 subunit rather than self associate is evident by the detection of the ,B2-B3-crystallin heterodimer by native gel electrophoresis and electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry under non denaturing conditions. The complex has been detected for the first time and its molecular mass is measured to be 47 450 ± 1 Da. Radical probe mass spectrometry (RP-MS) was subsequently applied to investigate the nature of the heterodimer through the limited oxidation of the subunits in the complex. Two peptide segments of the ,B2 subunit and six of the ,B3 subunit were found to oxidise, with far greater oxidation observed within the ,B3 versus the ,B2 subunit. This, and the observation that the oxidation data of ,B2 subunit is inconsistent with the structure of the ,B2 monomer, demonstrates that the protection of ,B2 is conferred by its association with ,B3 subunit within the heterodimer where only the residues of, and towards, its N -terminal domain remain exposed to solvent. The results suggest that the ,B2 subunit adopts a more compacted form than in its monomeric form in order for much of its structure to be enveloped by the ,B3 subunit within the heterodimer. Copyright © 2009 John Wiley & Sons, Ltd. [source] Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stabilityPROTEIN SCIENCE, Issue 9 2010Lynn Ziegler Abstract Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N-linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high-molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N , A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high-affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N-linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N-linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex. [source] Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDSPROTEIN SCIENCE, Issue 7 2006Junghwa Kim Abstract Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS,gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers. [source] Exploring the priming mechanism of liver regeneration: proteins and protein complexesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2009Xinyu Deng Abstract The liver has the ability to restore its functional capacity following injury or resection and the priming of liver regeneration is a complex process that has not been completely elucidated. In the current research, to further reveal the priming mechanism of liver regeneration, hepatocyte total protein and hepatocyte cytosol of the rats at 4,h after 2/3 partial hepatectomy (PHx) were studied, respectively, by 2-DE and 2-D blue native gel electrophoresis. Seventeen unique differential proteins were identified in hepatocyte total protein samples. Nine differential protein complexes containing 41 protein components were identified in hepatocyte cytosol samples. For the first time, at the priming stage of liver regeneration, the variations of serine protease inhibitor 2c, sulfite oxidase and valosin-containing protein (VCP) were presented and validated by Western blotting, and the VCP complex was further validated by antibody super-shift experiments. The current results suggested that at 4,h after PHx, VCP complex was down-regulated in hepatocyte cytosol, apoptosis pathways were inhibited, nuclear factor-,B and interleukin 6 pathways worked together and triggered the liver regeneration. [source] X-linked NDUFA1 gene mutations associated with mitochondrial encephalomyopathyANNALS OF NEUROLOGY, Issue 1 2007Daniel Fernandez-Moreira PharmB Objective Mitochondrial complex I deficiency is the commonest diagnosed respiratory chain defect, being genetically heterogeneous. The male preponderance of previous patient cohorts suggested an X-linked underlying genetic defect. We investigated mutations in the X-chromosomal complex I structural genes, NDUFA1 and NDUFB11, as a novel cause of mitochondrial encephalomyopathy. Methods We sequenced 12 nuclear genes and the mitochondrial DNA,encoded complex I genes in 26 patients with respiratory chain complex I defect. Novel mutations were confirmed by polymerase chain reaction restriction length polymorphism. Assembly/stability studies in fibroblasts were performed using two-dimensional blue native gel electrophoresis. Results Two novel p.Gly8Arg and p.Arg37Ser hemizygous mutations in NDUFA1 were identified in two unrelated male patients presenting with Leigh's syndrome and with myoclonic epilepsy and developmental delay, respectively. Two-dimensional blue native gel electrophoresis showed decreased levels of intact complex I with no accumulation of lower molecular weight subcomplexes, indicating that assembly, stability, or both are compromised. Interpretation Mutations in the X-linked NDUFA1 gene result in complex I defect and encephalomyopathy. Assembly/stability analysis might give an explanation for the different clinical phenotypes and become useful for future diagnostic purposes. Ann Neurol 2007;61:73,83 [source] The production, purification and crystallization of a soluble heterodimeric form of a highly selected T-cell receptor in its unliganded and liganded stateACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2002Craig S. Clements T-cell antigen receptors (TcRs) are heterodimeric cell-surface receptors that play a pivotal role in the cellular immune response. The TcR interacts specifically with a peptide-laden major histocompatability complex (pMHC). A human TcR has been characterized that interacts with an immunodominant epitope, FLRGRAYGL, from the Epstein,Barr virus, a ubiquitous human pathogen, in complex with HLA-B8. Despite the vast TcR repertoire, this TcR is found in up to 10% of the total T-cell population in seropositive HLA-B8+ individuals. In this report, this highly selected TcR is characterized by expressing in Escherichia coli, refolding, purifying and crystallizing the receptor. In addition, the HLA-B8,FLRGRAYGL complex has been expressed in E. coli, refolded and shown to be functionally active. Using native gel electrophoresis, the refolded TcR is shown to be capable of binding specifically to the refolded HLA-B8,FLRGRAYGL and this TcR has been crystallized in complex with the pMHC. The crystals of the unliganded and liganded TcR diffract to 1.5 and 2.5,Å, respectively. [source] | |||||||||||||