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

Research output: Contribution to journalArticle

Abstract

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha subunit with the NAD(H)-binding domain I and a beta 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 alpha and beta subunits. The interface in domain II between the alpha and the beta subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the alpha subunit and loops connecting the nine transmembrane helices in the beta subunit. However, to investigate the organization of the nine transmembrane helices in the beta 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 alpha subunit and the two new peptides beta1 and beta2. 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 alpha subunit was normally folded, followed by a concerted folding of beta1 + beta2. Cross-linking of a betaS105C-betaS237C 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 beta subunit has been demonstrated.

Details

Authors
  • Magnus Althage
  • Jenny Karlsson
  • Pontus Gourdon
  • Mikael Levin
  • Roslyn M Bill
  • Anna Tigerström
  • Jan Rydström
External organisations
  • University of Gothenburg
Research areas and keywords

Keywords

  • Catalysis, Codon, Cross-Linking Reagents, Cysteine, Electrophoresis, Polyacrylamide Gel, Escherichia coli, Factor Xa, Kinetics, Models, Biological, Mutagenesis, Mutagenesis, Site-Directed, Mutation, NAD, NADP, NADP Transhydrogenases, Peptides, Plasmids, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Proteolipids, Protons, Time Factors, Trypsin, Journal Article, Research Support, Non-U.S. Gov't
Original languageEnglish
Pages (from-to)10998-11003
JournalBiochemistry
Volume42
Issue number37
Publication statusPublished - 2003 Sep 23
Publication categoryResearch
Peer-reviewedYes
Externally publishedYes