Separation methods in the analysis of protein membrane complexes

Kashino, Yasuhiro

doi:10.1016/s1570-0232(03)00428-8

Cited by 76 publications

(82 citation statements)

References 174 publications

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“…The observed molecular mass for WecA FLAG-5ϫHis was lower than the predicted mass deduced from its amino acid sequence (44.8 kDa). The anomalous migration of WecA in SDS-PAGE was reported previously (3-5) and was probably due to the hydrophobicity and high pI of this protein (theoretical pI, 10.01) (20). The bands at higher molecular masses in the blot were likely due to WecA FLAG-5ϫHis oligomers that resulted from the mild denaturation conditions (incubation at 45°C for 30 min in loading sample buffer containing 50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, and 0.1% bromophenol blue) used to visualize WecA since, as we previously reported (5) higher temperatures resulted in a failure to detect WecA.…”

Section: Resultssupporting

confidence: 79%

Functional Characterization and Membrane Topology of Escherichia coli WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide

et al. 2007

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WecA is an integral membrane protein that initiates the biosynthesis of enterobacterial common antigen and O-antigen lipopolysaccharide (LPS) by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate (Und-P) to form Und-P-P-GlcNAc. WecA belongs to a large family of eukaryotic and prokaryotic prenyl sugar transferases. Conserved aspartic acids in putative cytoplasmic loops 2 (Asp90 and Asp91) and 3 (Asp156 and Asp159) were targeted for replacement mutagenesis with either glutamic acid or asparagine. We examined the ability of each mutant protein to complement O-antigen LPS synthesis in a wecA-deficient strain and also determined the steady-state kinetic parameters of the mutant proteins in an in vitro transfer assay. Apparent K m and V max values for UDP-GlcNAc, Mg 2؉ , and Mn 2؉ suggest that Asp156 is required for catalysis, while Asp91 appears to interact preferentially with Mg 2؉ , possibly playing a role in orienting the substrates. Topological analysis using the substituted cysteine accessibility method demonstrated the cytosolic location of Asp90, Asp91, and Asp156 and provided a more refined overall topological map of WecA. Also, we show that cells expressing a WecA derivative C terminally fused with the green fluorescent protein exhibited a punctate distribution of fluorescence on the bacterial surface, suggesting that WecA localizes to discrete regions in the bacterial plasma membrane.

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Section: Resultssupporting

confidence: 79%

Functional Characterization and Membrane Topology of Escherichia coli WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide

et al. 2007

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“…9a, lane 2, white arrow), which was interpreted as a WbaP M170-R354-FLAG degradation product. Protein aggregates that barely penetrate the gel matrix and anomalous migration in the gel are commonly observed with membrane proteins upon heat denaturation, particularly in proteins with high pIs (Kashino, 2003). To avoid this problem, the protein samples were incubated at 45 u C for 30 min, as we have done in previous studies with the integral membrane proteins WecA and Wzx (Amer & Valvano, 2000;Lehrer et al, 2007;Marolda et al, 2004).…”

Section: Expression Of Wbap Protein Derivatives As Flag Epitope Fusionsmentioning

confidence: 99%

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

et al. 2008

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WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in DwbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the DwbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.

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“…2A. As is often the case with strongly hydrophobic membrane proteins, 23) boiling of protein samples in SDS loading buffer prior on SDS-PAGE often results in significant aggregation of Gtb PAP2L2s so that they do not enter the resolving gel. Protein samples were therefore incubated in SDS loading buffer at room temperature before gel loading.…”

Section: Pap2l2 Proteinmentioning

confidence: 99%

Cloning, Expression, and Characterization of a Thermostable PAP2L2, a New Member of the Type-2 Phosphatidic Acid Phosphatase Family fromGeobacillus toebiiT-85

Zhang

Yang

Huang

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Separation methods in the analysis of protein membrane complexes

Cited by 76 publications

References 174 publications

Functional Characterization and Membrane Topology of Escherichia coli WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide

Functional Characterization and Membrane Topology of Escherichia coli WecA, a Sugar-Phosphate Transferase Initiating the Biosynthesis of Enterobacterial Common Antigen and O-Antigen Lipopolysaccharide

Distinct functional domains of the Salmonella enterica WbaP transferase that is involved in the initiation reaction for synthesis of the O antigen subunit

Cloning, Expression, and Characterization of a Thermostable PAP2L2, a New Member of the Type-2 Phosphatidic Acid Phosphatase Family fromGeobacillus toebiiT-85

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