Althage Magnus, Karlsson Jenny, Gourdon Pontus, Levin Mikael, Bill Roslyn M, Tigerström Anna, Rydström Jan
Department of Biochemistry and Biophysics, Göteborg University, Sweden.
Biochemistry. 2003 Sep 23;42(37):10998-1003. doi: 10.1021/bi034560x.
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.
来自大肠杆菌的质子泵烟酰胺核苷酸转氢酶包含一个带有NAD(H)结合结构域I的α亚基和一个带有NADP(H)结合结构域III的β亚基。膜结构域（结构域II）包含质子通道，由α亚基和β亚基的疏水部分组成。先前已通过交联连接α亚基中四个跨膜螺旋的环和连接β亚基中九个跨膜螺旋的环，对结构域II中α亚基和β亚基之间的界面进行了研究。然而，为了研究β亚基中九个跨膜螺旋的组织情况，通过在连接跨膜螺旋9和10的环中利用现有的下游起始密码子通过单一诱变步骤创建一个终止密码子，引入了一个分裂。所得的酶由野生型α亚基以及两个新的肽β1和β2组成。与其他分裂膜蛋白相比，新的转氢酶具有显著的活性，能够催化NADPH还原3-乙酰吡啶-NAD(+)、NADH（由结合的NADP(H)介导）循环还原3-乙酰吡啶-NAD(+)以及质子泵作用，其活性相当于相应野生型活性的约50-107%。这些高活性表明α亚基正常折叠，随后β1 + β2协同折叠。在无半胱氨酸的功能性分裂背景下对βS105C-βS237C双半胱氨酸突变体进行交联，然后进行SDS-PAGE分析，结果表明螺旋9、13和14彼此靠近。这是首次证明同一β亚基中螺旋之间的交联。
