(582df) Evaluating a Novel Aquaporin From R. Sphaeroides for Biomimetic Membrane Applications

We demonstrate that a novel aquaporin from Rhodobacter sphaeroides 2.4.1., RsAqpZ, displays superior water transport characteristics in phospholipid vesicles as compared to well-studied E.coli AqpZ and most of the other orthodox aquaporins.

BACKGROUND:Most biological cells are surrounded by phospholipid bilayer protecting them from their surroundings and compartmentalizing subcellular organelles to carry out particular processes. Selective permeable nature of the lipid bilayer allows certain organic molecules and ions across the cell with high efficiency and selectivity, which is essential for maintenance of the cellular homeostasis. Membrane proteins are the key players in recognition and transportation of the solutes with a low energy input and excellent specificity. These exceptional features of membrane proteins inspired many researchers from a variety of disciplines to solve difficult separation problems in sensor technology, energy generation and seawater desalination. Aquaporins, one particular family of highly selective water channel proteins integrated in plasma membrane of a wide spectrum of biological cells ranging from bacteria to mammalian cells, emerged as a great potential for water desalination industries. Aquaporins facilitate the high flux of H₂O merely depending on osmotic pressure gradient across the cell membrane without consuming cellular biochemical energy, while they reject ions and organic molecules. High water transport rate and specificity of aquaporins make them potential candidates for utilization in next generation water desalination membranes to reduce the high operational costs required by reverse osmosis membranes in seawater desalination and purification. Since Rhodobacter species live in diverse environments, such as consuming organic acids under anaerobic photosynthetic conditions, cloning, expression and characterization of a novel aquaporin from Rhodobacter sphaeroides 2.4.1., RsAqpZ, was undertaken in anticipation of obtaining a more robust aquaporin that is amenable to biomimetic membrane applications with high stability and enhanced water permeability.

RESULTS: RsAqpZ was expressed in Rhodobacter under anaerobic photoheterotrophic growth conditions with a yield reaching at 30 mg/L in its homologous expression system. We compared water transport characteristics of RsAqpZ with widely used E.coli AqpZ (EcAqpZ) running stopped flow-light scattering experiments using phospholipid vesicles reconstituted with these proteins. A higher water permeability (0.1700 ± 0.0500 cm/s) and lower energy of activation for water transport (2.93 ± 0.60 kcal/mol) was measured for RsAqpZ. Efficiency of incorporation was also significantly higher for RsAqpZ, as we observed a linear relationship between water permeability and protein mass fraction in proteoliposomes until a protein/lipid ratio of 0.04. The highest protein/lipid ratio, we could obtain with EcAqpZ remained to be 0.01. Initial investigation of incorporation in triblock copolymers resulted a similar trend, as well, which indicates that can be RsAqpZ a good alternative for EcAqpZ for utilization in biomimetic water desalination membranes. For a better understanding of the enhanced incorporation ability of RsAqpZ, we performed 2D crystallization tests with DOPC lipids using microdialysis method. Initial results predicted a tetragonal symmetry for RsAqpZ in phospholipid membranes showing promises for structure determination by electron crystallography. Finally, we constructed fluorescent protein fusions of RsAqpZ to estimate the single channel permeability using a new method based on fluorescence correlation spectroscopy. The presence of the fluorescent protein tag did not change the expression level and water permeability of RsAqpZ as evidenced by Western-blot analysis and stopped-flow light scattering, respectively.

CONCLUSION: A novel water channel protein from purple bacterium Rhodobacter sphaeroides, RsAqpZ was cloned, overexpressed and purified in functional form. Evaluation of functionality demonstrated high water permeability, low activation energy and high incorporation efficiency in liposome and polymersome systems. Our findings suggest that RsAqpZ might be a better alternative to EcAqpZ for utilization in biomimetic water desalination membranes.