Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli

Magnus Althage, Jenny Karlsson, Pontus Gourdon, Mikael Levin, Roslyn M. Bill, Anna Tigerström, Jan Rydström

Research output: Contribution to journalArticle

Abstract

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α subunit with the NAD(H)-binding domain I and a β subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the α and β subunits. The interface in domain II between the α and the β subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the α subunit and loops connecting the nine transmembrane helices in the β subunit. However, to investigate the organization of the nine transmembrane helices in the β subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type α subunit and the two new peptides β1 and β2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD + by NADPH, the cyclic reduction of 3-acetylpyridine-NAD + by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the α subunit was normally folded, followed by a concerted folding of β1 + β2. Cross-linking of a βS105C-βS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same β subunit has been demonstrated.

Original languageEnglish
Pages (from-to)10998-11003
Number of pages6
JournalBiochemistry
Volume42
Issue number37
DOIs
Publication statusPublished - 23 Sep 2003

Fingerprint

NAD
Crosslinking
Escherichia coli
Protons
NADP
Membranes
Cysteine
NADP Transhydrogenases
Mutagenesis
Initiator Codon
Terminator Codon
Ports and harbors
Polyacrylamide Gel Electrophoresis
Membrane Proteins
Peptides
Enzymes
3-acetylpyridine

Keywords

  • proton pumping nicotinamide nucleotide transhydrogenase
  • escherichia coli
  • subunit
  • membrane domain
  • proton channel

Cite this

Althage, Magnus ; Karlsson, Jenny ; Gourdon, Pontus ; Levin, Mikael ; Bill, Roslyn M. ; Tigerström, Anna ; Rydström, Jan. / Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli. In: Biochemistry. 2003 ; Vol. 42, No. 37. pp. 10998-11003.
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abstract = "Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α subunit with the NAD(H)-binding domain I and a β subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the α and β subunits. The interface in domain II between the α and the β subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the α subunit and loops connecting the nine transmembrane helices in the β subunit. However, to investigate the organization of the nine transmembrane helices in the β subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type α subunit and the two new peptides β1 and β2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD + by NADPH, the cyclic reduction of 3-acetylpyridine-NAD + by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107{\%} of the corresponding wild-type activities. These high activities suggest that the α subunit was normally folded, followed by a concerted folding of β1 + β2. Cross-linking of a βS105C-βS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same β subunit has been demonstrated.",
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Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli. / Althage, Magnus; Karlsson, Jenny; Gourdon, Pontus; Levin, Mikael; Bill, Roslyn M.; Tigerström, Anna; Rydström, Jan.

In: Biochemistry, Vol. 42, No. 37, 23.09.2003, p. 10998-11003.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli

AU - Althage, Magnus

AU - Karlsson, Jenny

AU - Gourdon, Pontus

AU - Levin, Mikael

AU - Bill, Roslyn M.

AU - Tigerström, Anna

AU - Rydström, Jan

PY - 2003/9/23

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KW - proton pumping nicotinamide nucleotide transhydrogenase

KW - escherichia coli

KW - subunit

KW - membrane domain

KW - proton channel

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