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

PLoS One. 2014 Jan 31;9(1):e86830. doi: 10.1371/journal.pone.0086830. eCollection 2014.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Amino Acid Sequence
  • Aquaporins / classification
  • Aquaporins / genetics
  • Aquaporins / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Biological Transport
  • Blotting, Western
  • Cell Membrane Permeability
  • Cloning, Molecular
  • Gene Expression Regulation, Bacterial
  • Liposomes / metabolism
  • Liposomes / ultrastructure
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Electron
  • Models, Biological
  • Molecular Sequence Data
  • Phylogeny
  • Recombinant Proteins / metabolism*
  • Rhodobacter sphaeroides / genetics
  • Rhodobacter sphaeroides / metabolism*
  • Sequence Homology, Amino Acid
  • Water / metabolism

Substances

  • Aquaporins
  • Bacterial Proteins
  • Liposomes
  • Luminescent Proteins
  • Recombinant Proteins
  • Water

Grants and funding

Grace-Woodward Foundation provided support for development of Rhodobacter as a membrane protein expression platform, which set the basis for this work. ARPA-E’s support helped development of genetic transformation methods for Rhodobacter. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.