Molecular cloning, overexpression and characterization of a novel water channel protein from Rhodobacter sphaeroides

• Published in: PloS one Vol. 9; no. 1; p. e86830
• Main Authors: Erbakan, Mustafa, Shen, Yue-xiao, Grzelakowski, Mariusz, Butler, Peter J, Kumar, Manish, Curtis, Wayne R
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 31.01.2014; Public Library of Science (PLoS)
• Subjects: Activation energy; Algorithms; Amino Acid Sequence; Amino acids; Anaerobic conditions; Aquaporins; Aquaporins - classification; Aquaporins - genetics; Aquaporins - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biochemistry; Biological Transport; Biology; Biomedical engineering; Block copolymers; Blood; Blood cells; Blotting, Western; Cell culture; Cell Membrane Permeability; Chemical engineering; Chemistry; Cloning; Cloning, Molecular; Copolymers; E coli; Erythrocytes; Eye lens; Fluorescence; Fluorescence spectroscopy; Fusion protein; Gene Expression Regulation, Bacterial; Genetic aspects; Glycerol; Growth conditions; Homology; Kidneys; Light scattering; Lipids; Liposomes - metabolism; Liposomes - ultrastructure; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Materials Science; Measurement techniques; Membranes; Microorganisms; Microscopy; Microscopy, Electron; Models, Biological; Molecular Sequence Data; Organisms; Permeability; Photosynthesis; Phylogeny; Plant roots; Plasma membranes; Proteins; Recombinant Proteins - metabolism; Rhodobacter sphaeroides; Rhodobacter sphaeroides - genetics; Rhodobacter sphaeroides - metabolism; Sequence Homology, Amino Acid; Spectroscopy; Studies; Vertebrates; Water - metabolism; Water purification; Water transport; Water treatment; United States > US; Pennsylvania
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0086830

Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport (2.93±0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10(-12) cm(3)/s or 4.17 (±1.38)×10(10) H2O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes.