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

TIGERSTRÖM, ANNA KATARINA, 1976 (författare)
Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
Karlsson, J (författare)
Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
Gourdon, Pontus (författare)
Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
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Levin, M (författare)
Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
Bill, Roslyn M. (författare)
Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
Tigerström, Anna, 1976 (författare)
Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry
Rydström, Jan, 1943 (författare)
Gothenburg University,Göteborgs universitet,Institutionen för kemi,Department of Chemistry,University of Gothenburg
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 (creator_code:org_t)
2003-08-26
2003
Engelska.
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 42, s. 10998-11003
  • Tidskriftsartikel (refereegranskat)
Abstract Ämnesord
Stäng  
  • 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.

Ämnesord

NATURVETENSKAP  -- Kemi (hsv//swe)
NATURAL SCIENCES  -- Chemical Sciences (hsv//eng)

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