Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

Background: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-beta-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly a-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58 degrees C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70 degrees C. Conclusion/Significance: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications

Established and proposed roles of xanthine oxidoreductase in oxidative and reductive pathways in plants

Xanthine oxidoreductase (XOR) is among the most-intensively studied enzymes known to participate in the consumption of oxygen in cells. However, it attracted the attention of researchers due its participation in free radical production in vivo, mainly through the production of superoxide radicals. In plants, XOR is a key enzyme in purine degradation where it catalyzes the oxidation of hypoxanthine to xanthine and of xanthine to uric acid. Both reactions are accompanied by electron transfer to either NAD+ with simultaneous formation of NADH or to molecular oxygen, which results in formation of superoxides. Characterization of plant XOR mutants and isolated XOR proteins from various plant species provided evidence that the enzyme plays significant roles in plant growth, leaf senescence, fruit size, synthesis of nitrogen storage compounds, and plant-pathogen interactions. Moreover, the ability of XOR to carry out redox reactions as NADH oxidase and to produce reactive oxygen species and nitric oxide, together with a possible complementary role in abscisic acid synthesis have raised further attention on the importance of this enzyme. Based on these established and proposed functions, XOR is discussed as regulator of different processes of interest in plant biology and agriculture.The authors acknowledge the support of the research grants AGL2010-16167 to J.F.M. from the Spanish Ministry of Science and Innovation and Bi 1075/5-1 to F.B. by the Deutsche Forschungsgemeinschaft. R.E. received a JAE-Doctor grant from the Spanish Research Council (CSIC).