Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

Structure of the Functionally Important Extracellular Loop C of Human Aquaporin 1 Obtained by Solid-State NMR under Nearly Physiological Conditions.

Human aquaporin 1 (hAQP1) is the first discovered selective water channel present in lipid membranes of multiple types of cells. Several structures of hAQP1 and its bovine homolog have been obtained by electron microscopy and X-ray crystallography, giving a consistent picture of the transmembrane domain with the water-conducting pore. The transmembrane domain is formed by six full helices and two half-helices, which form a central constriction with conserved asparagine-proline-alanine motifs. Another constriction, the aromatic/arginine (ar/R) filter, is found close to the extracellular surface, and includes aromatic residues and a conserved arginine (Arg-195). Although the existing crystal structures largely converge on the location of helical segments, they differ in details of conformation of the longest extracellular loop C and its interactions with the ar/R filter (in particular, with Arg-195). Here, we use solid-state nuclear magnetic resonance to determine multiple interatomic distances, and come up with a refined structural model for hAQP1, which represents a physiologically relevant state predominant at noncryogenic temperatures in a lipid environment. The model clearly disambiguates the position of the Arg-195 sidechain disputed previously and shows a number of interactions for loop C, both with the ar/R filter and a number of other residues on the extracellular side of hAQP1.

Water recycling at the Nova Scotia Hospital Central Laundry.

A water recycling system was installed at the Nova Scotia Hospital Central Laundry in Dartmouth, Nova Scotia in the spring of 2014. The consequent reductions in the consumption of water and steam at this facility have been measured over a 20-month period and are presented here in nominal units and as a financial analysis. Cumulative savings over this period have been calculated to be $351,567, representing a 53% annual return on investment after accounting for annual operating and maintenance costs.