Crystallization of Human Aquaglyceroporins for Neutron Diffraction Studies

Abstract

Aquaglyceroporins are channels that facilitate the flux of glycerol and water across lipid bilayers. Although structural information is available for several aquaglyceroporins, the details of how water and glycerol selectivity are maintained and how protons are excluded remain elusive. An approach to obtaining data on the hydrogen atom positions is to apply neutron macromolecular crystallography. Here, we present strategies to obtain large crystals suitable for neutron diffraction experiments by assessing a range of different methods, including new procedures for protein purification and crystallization. By applying long incubation times, macroseeding, and/or optimization of detergents, millimeter-sized crystals with different morphologies were obtained, and their diffraction quality was assessed by exposure to X-rays. The data presented here lay the foundation for continued crystallization efforts targeting aquaporins and other membrane proteins for neutron diffraction experiments.