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

Plasencia I, Survery S, Ibragimova S, Hansen JS, Kjellbom P, Helix-Nielsen C, Johanson U, Mouritsen OG - PLoS ONE (2011)

Bottom Line: SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields.Generally, SoPIP2;1 secondary structure was found to be predominantly α-helical in accordance with crystallographic data.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Chemistry, MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark. miplsen@memphys.sdu.dk

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-β-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 α-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.

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MRE at 222 nm as a function of temperature.SoPIP2;1 in OG micelles (•), E. coli lipid membranes (□), POPE∶POPC∶POPS∶Ergosterol ( o) and POPE∶POPC (◊).
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pone-0014674-g003: MRE at 222 nm as a function of temperature.SoPIP2;1 in OG micelles (•), E. coli lipid membranes (□), POPE∶POPC∶POPS∶Ergosterol ( o) and POPE∶POPC (◊).

Mentions: A summary of the results can be found in Figure 3 for the thermal stability of the secondary structure where the Mean residue ellipticity (MRE) at 222 nm were plotted as a function of the temperature for all systems studied. The different MRE values at 20°C (initial state) demonstrate that the protein is structured or interacting in different ways with the different detergents or lipids used. Therefore it can be assumed that the protein acquires different structural initial states as quantified by the deconvolution analyses in the different environments tested (Table 1). The SoPIP2;1 structure is more sensitive to the temperature changes when it is reconstituted into detergent micelles than when it is reconstituted into lipid membrane systems. Although the unfolding transition is not reversible, the transition midpoint can be used to quantify the thermal stability of the protein [33].


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

Plasencia I, Survery S, Ibragimova S, Hansen JS, Kjellbom P, Helix-Nielsen C, Johanson U, Mouritsen OG - PLoS ONE (2011)

MRE at 222 nm as a function of temperature.SoPIP2;1 in OG micelles (•), E. coli lipid membranes (□), POPE∶POPC∶POPS∶Ergosterol ( o) and POPE∶POPC (◊).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3038850&req=5

pone-0014674-g003: MRE at 222 nm as a function of temperature.SoPIP2;1 in OG micelles (•), E. coli lipid membranes (□), POPE∶POPC∶POPS∶Ergosterol ( o) and POPE∶POPC (◊).
Mentions: A summary of the results can be found in Figure 3 for the thermal stability of the secondary structure where the Mean residue ellipticity (MRE) at 222 nm were plotted as a function of the temperature for all systems studied. The different MRE values at 20°C (initial state) demonstrate that the protein is structured or interacting in different ways with the different detergents or lipids used. Therefore it can be assumed that the protein acquires different structural initial states as quantified by the deconvolution analyses in the different environments tested (Table 1). The SoPIP2;1 structure is more sensitive to the temperature changes when it is reconstituted into detergent micelles than when it is reconstituted into lipid membrane systems. Although the unfolding transition is not reversible, the transition midpoint can be used to quantify the thermal stability of the protein [33].

Bottom Line: SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields.Generally, SoPIP2;1 secondary structure was found to be predominantly α-helical in accordance with crystallographic data.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Chemistry, MEMPHYS-Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark. miplsen@memphys.sdu.dk

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-β-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 α-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.

Show MeSH