Organization in the Membrane of the N-Terminal Proton-Translocating Domain of the β Subunit of the Pyridine Nucleotide Transhydrogenase of Escherichia coli

• Published in: Biochemical and biophysical research communications 1995-09, Vol.214 (1), p.230-238
• Main Authors: Glavas, N.A., Hou, C., Bragg, P.D.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Cell Membrane - enzymology; Cysteine - chemistry; Escherichia coli - enzymology; Ion Transport; Molecular Sequence Data; Mutagenesis, Site-Directed; NADP - metabolism; NADP Transhydrogenases - chemistry; NADP Transhydrogenases - genetics; NADP Transhydrogenases - metabolism; Protons; Sulfhydryl Reagents
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1006/bbrc.1995.2279

The proton-translocating transmembrane pyridine nucleotide transhydrogenase of Escherichia coli is composed of two types of subunits, α and β, The β subunit has several membrane-spanning segments in the N-terminal region followed by a cytosolic C-terminal domain bearing a binding site for NADP(H), The N-terminal region contains at least one residue involved in the process of transmembrane proton translocation. Using site-directed mutagenesis cysteine residues were introduced at selected sites into the N-terminal region of the β subunit. The pattern of labelling of these residues with 3-(N-maleimidyl propionyl)biocytin and other sulfhydryl reagents has shown that a model in which the N-terminal region of the β subunit spans the membrane in eight segments is more likely than a previously proposed six segment model (Holmberg et al. (1994) Biochemistry 33, 7691-7700), The preferred model accounts for the site of labelling of a glutamate residue (Glu124) in the N-terminal domain by N,N′-dicyclohexyicarbodiimide.