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

Althage, Magnus; Karlsson, Jenny; Gourdon, Pontus LU ; Levin, Mikael; Bill, Roslyn M; Tigerström, Anna and Rydström, Jan (2003) In Biochemistry 42(37). p.10998-11003
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... (More)

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.

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author
publishing date
type
Contribution to journal
publication status
published
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
in
Biochemistry
volume
42
issue
37
pages
10998 - 11003
publisher
The American Chemical Society
external identifiers
  • scopus:0141431037
ISSN
0006-2960
DOI
10.1021/bi034560x
language
English
LU publication?
no
id
062736b1-febc-4fd6-9cde-3fdcc6fb763d
date added to LUP
2017-04-29 15:33:06
date last changed
2017-05-07 04:52:52
@article{062736b1-febc-4fd6-9cde-3fdcc6fb763d,
  abstract     = {<p>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.</p>},
  author       = {Althage, Magnus and Karlsson, Jenny and Gourdon, Pontus and Levin, Mikael and Bill, Roslyn M and Tigerström, Anna and Rydström, Jan},
  issn         = {0006-2960},
  keyword      = {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},
  language     = {eng},
  month        = {09},
  number       = {37},
  pages        = {10998--11003},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Functional split and crosslinking of the membrane domain of the beta subunit of proton-translocating transhydrogenase from Escherichia coli},
  url          = {http://dx.doi.org/10.1021/bi034560x},
  volume       = {42},
  year         = {2003},
}