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

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α subunit with the NAD(H)-binding domain I and a β 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 α and β subunits. The interface in domain II between the α and the β subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the α subunit and loops connecting the nine transmembrane helices in the β subunit. However, to investigate the organization of the nine transmembrane helices in the β 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 α subunit and the two new peptides β1 and β2. 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 α subunit was normally folded, followed by a concerted folding of β1 + β2. Cross-linking of a βS105C-βS237C 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 β subunit has been demonstrated.

Abercrombie and others v Aga Rangemaster 2012

Case law report - online

F and G Cleaners Ltd v Saddington 2012

Case law report - online

Political performance and leadership persona:the UK Labour Party Conference of 2012

This article is a contribution to an emerging scholarship on the role of rhetoric, persona and celebrity, and the effects of performance on the political process. We analyse party leader Ed Miliband at the UK Labour Party Conference in Manchester in 2012. Our analysis identifies how, through performance of himself and the beginnings of the deployment of an alternative party narrative centred on One Nation, Ed Miliband began to revise his received persona. By using a range of rhetorical and other techniques, Miliband began to adapt the Labour narrative to the personalized political. The article sets out the theoretical framework for the analysis and returns to the implications for the theory of leadership performance in its conclusion.