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Topology and accessibility of the transmembrane helices and the sensorysite in the bifunctional transporter DcuB of escherichia coil

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Topology and accessibility of the transmembrane helices and the sensorysite in the bifunctional transporter DcuB of escherichia coil. / Bauer, Julia; Fritsch, Maximilian; Palmer, Tracy; Unden, Gottfried.

In: Biochemistry, Vol. 50, No. 26, 05.07.2011, p. 5925-5938.

Research output: Contribution to journalArticle

Harvard

Bauer, J, Fritsch, M, Palmer, T & Unden, G 2011, 'Topology and accessibility of the transmembrane helices and the sensorysite in the bifunctional transporter DcuB of escherichia coil' Biochemistry, vol 50, no. 26, pp. 5925-5938.

APA

Bauer, J., Fritsch, M., Palmer, T., & Unden, G. (2011). Topology and accessibility of the transmembrane helices and the sensorysite in the bifunctional transporter DcuB of escherichia coil. Biochemistry, 50(26), 5925-5938doi: 10.1021/bi1019995

Vancouver

Bauer J, Fritsch M, Palmer T, Unden G. Topology and accessibility of the transmembrane helices and the sensorysite in the bifunctional transporter DcuB of escherichia coil. Biochemistry. 2011 Jul 5;50(26):5925-5938.

Author

Bauer, Julia; Fritsch, Maximilian; Palmer, Tracy; Unden, Gottfried / Topology and accessibility of the transmembrane helices and the sensorysite in the bifunctional transporter DcuB of escherichia coil.

In: Biochemistry, Vol. 50, No. 26, 05.07.2011, p. 5925-5938.

Research output: Contribution to journalArticle

Bibtex - Download

@article{131139f3b46c44fdb7c5d68202e4fc07,
title = "Topology and accessibility of the transmembrane helices and the sensorysite in the bifunctional transporter DcuB of escherichia coil",
author = "Julia Bauer and Maximilian Fritsch and Tracy Palmer and Gottfried Unden",
year = "2011",
volume = "50",
number = "26",
pages = "5925--5938",
journal = "Biochemistry",
issn = "0006-2960",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - Topology and accessibility of the transmembrane helices and the sensorysite in the bifunctional transporter DcuB of escherichia coil

A1 - Bauer,Julia

A1 - Fritsch,Maximilian

A1 - Palmer,Tracy

A1 - Unden,Gottfried

AU - Bauer,Julia

AU - Fritsch,Maximilian

AU - Palmer,Tracy

AU - Unden,Gottfried

PY - 2011/7/5

Y1 - 2011/7/5

N2 - <p>C-4-Dicarboxylate uptake transporter B (DcuB) of Escherichia coil is a bifunctional transporter that catalyzes fumarate/succinate antiport and serves as a cosensor of the sensor kinase DcuS. Sites and domains of DcuB were analyzed for their topology relative to the cytoplasmic or periplasmic side of the membrane and their accessibility to the water space. For the topology studies, DcuB was fused at 33 sites to the reporter enzymes PhoA and LacZ that are only active when located in the periplasm or the cytoplasm, respectively. The ratios of the PhoA and LacZ activities suggested the presence of 10 or 11 hydrophilic loops, and 11 or 12 alpha-helical transmembrane domains (TMDs). The central part of DcuB allowed no clear topology prediction with LacZ/PhoA fusions. The sites of DcuB accessible to the hydrophilic thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate (AMS) were determined with variants of DcuB that carried single Cys residues. After intact cells were labeled with the membrane-impermeable AMS, denatured cells were differentially labeled with the thiol reagent polyethylene-glycol-maleimide (PEGmal) and analyzed for a mass shift. From 35 positions 17 were accessible to AMS in intact bacteria. The model derived from topology and accessibility suggests 12 TMDs for DcuB and a waterfilled cavity in its central part. The cavity ends with a cytoplasmic lid accessible to AMS from the periplasmic side. The sensory domain of DcuB is composed of cytoplasmic loop XI/XII and a membrane integral region with the regulatory residues Thr396/Asp398 and Lys353.</p>

AB - <p>C-4-Dicarboxylate uptake transporter B (DcuB) of Escherichia coil is a bifunctional transporter that catalyzes fumarate/succinate antiport and serves as a cosensor of the sensor kinase DcuS. Sites and domains of DcuB were analyzed for their topology relative to the cytoplasmic or periplasmic side of the membrane and their accessibility to the water space. For the topology studies, DcuB was fused at 33 sites to the reporter enzymes PhoA and LacZ that are only active when located in the periplasm or the cytoplasm, respectively. The ratios of the PhoA and LacZ activities suggested the presence of 10 or 11 hydrophilic loops, and 11 or 12 alpha-helical transmembrane domains (TMDs). The central part of DcuB allowed no clear topology prediction with LacZ/PhoA fusions. The sites of DcuB accessible to the hydrophilic thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate (AMS) were determined with variants of DcuB that carried single Cys residues. After intact cells were labeled with the membrane-impermeable AMS, denatured cells were differentially labeled with the thiol reagent polyethylene-glycol-maleimide (PEGmal) and analyzed for a mass shift. From 35 positions 17 were accessible to AMS in intact bacteria. The model derived from topology and accessibility suggests 12 TMDs for DcuB and a waterfilled cavity in its central part. The cavity ends with a cytoplasmic lid accessible to AMS from the periplasmic side. The sensory domain of DcuB is composed of cytoplasmic loop XI/XII and a membrane integral region with the regulatory residues Thr396/Asp398 and Lys353.</p>

KW - KINASE DCUS

KW - GENE-EXPRESSION

KW - C-4-DICARBOXYLATE TRANSPORT

KW - DICARBOXYLATE TRANSPORT

KW - MEMBRANE-PROTEINS

KW - HISTIDINE KINASES

KW - BINDING-SITE

KW - COLI

KW - MECHANISM

KW - BACTERIA

U2 - 10.1021/bi1019995

DO - 10.1021/bi1019995

M1 - Article

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 26

VL - 50

SP - 5925

EP - 5938

ER -

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