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Structure and stability of the spinach aquaporin SoPIP2;1 in detergent micelles and lipid membranes.

ABSTRACT: 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-?-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-2oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly ?-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°C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70°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.

SUBMITTER: Plasencia I

PROVIDER: S-EPMC3038850 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Structure and stability of the spinach aquaporin SoPIP2;1 in detergent micelles and lipid membranes.

Plasencia Inés I Survery Sabeen S Ibragimova Sania S Hansen Jesper S JS Kjellbom Per P Helix-Nielsen Claus C Johanson Urban U Mouritsen Ole G OG

PloS one 20110214 2

<h4>Background</h4>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.<h4>Methodology/principal finding</h4>We have characteri ...[more]

PMID: 21339815

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Project description:Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB(2), and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB(2), here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB(2) were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of (2)H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB(2) in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB(2) reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.

Dataset Information

Structure and stability of the spinach aquaporin SoPIP2;1 in detergent micelles and lipid membranes.

Publications

Structure and stability of the spinach aquaporin SoPIP2;1 in detergent micelles and lipid membranes.

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