Functional Split and Crosslinking of the Membrane Domain of the Subunit of Proton-Translocating Transhydrogenase fro
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- 作者：Magnus Althage ; Jenny Karlsson ; Pontus Gourdon ; Mikael Levin ; Roslyn M. Bill ; Anna Tigerströ ; m ; and Jan Rydströ ; m
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：September 23, 2003
- 年：2003
- 卷：42
- 期：37
- 页码：10998 - 11003
- 全文大小：99K
- 年卷期：v.42,no.37(September 23, 2003)
- ISSN：1520-4995
文摘
Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an
subunit with the NAD(H)-binding domain I and a 
subunit with the NADP(H)-binding domain III.The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic partsof the and subunits. The interface in domain II between the and the subunits has previously beeninvestigated by cross-linking loops connecting the four transmembrane helices in the subunit and loopsconnecting the nine transmembrane helices in the subunit. However, to investigate the organization ofthe nine transmembrane helices in the subunit, a split was introduced by creating a stop codon in theloop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existingdownstream start codon. The resulting enzyme was composed of the wild-type subunit and the twonew peptides 1 and 2. As compared to other split membrane proteins, the new transhydrogenase wasremarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD+ by NADPH, thecyclic 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 suggestthat the subunit was normally folded, followed by a concerted folding of 1 + 2. Cross-linking of aS105C-S237C double cysteine mutant in the functional split cysteine-free background, followed bySDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first timethat cross-linking between helices in the same subunit has been demonstrated.
subunit with the NAD(H)-binding domain I and a subunit with the NADP(H)-binding domain III.The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic partsof the and subunits. The interface in domain II between the and the subunits has previously beeninvestigated by cross-linking loops connecting the four transmembrane helices in the subunit and loopsconnecting the nine transmembrane helices in the subunit. However, to investigate the organization ofthe nine transmembrane helices in the subunit, a split was introduced by creating a stop codon in theloop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existingdownstream start codon. The resulting enzyme was composed of the wild-type subunit and the twonew peptides 1 and 2. As compared to other split membrane proteins, the new transhydrogenase wasremarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD+ by NADPH, thecyclic 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 suggestthat the subunit was normally folded, followed by a concerted folding of 1 + 2. Cross-linking of aS105C-S237C double cysteine mutant in the functional split cysteine-free background, followed bySDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first timethat cross-linking between helices in the same subunit has been demonstrated.