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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.

Show MeSH
Fluorescence intensity at the maximum of the emission spectra as a function of temperature.The protein reconstituted in detergent micelles show one principal transitions in comparison with the protein reconstituted in lipid membranes where at least three different transitions can be observed. Fluorescence intensity values are normalized to the higher intensity value observed with the protein in micelles suspension at 20°C.
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pone-0014674-g006: Fluorescence intensity at the maximum of the emission spectra as a function of temperature.The protein reconstituted in detergent micelles show one principal transitions in comparison with the protein reconstituted in lipid membranes where at least three different transitions can be observed. Fluorescence intensity values are normalized to the higher intensity value observed with the protein in micelles suspension at 20°C.

Mentions: SoPIP2;1 contains six tryptophan (Trp) residues in the primary sequence. These residues can be used as intrinsic fluorophores for analyzing the protein ternary structure (see the cartoon in Figure 5A) as the Trp fluorescence emission spectrum is sensitive to both the polarity and the dynamics of the environment surrounding the aromatic side chain. Therefore, the variation in the Trp fluorescence emission spectra reports changes in tertiary structure of the protein. Thus the Trp is generally blue-shifted from 350 nm in environments of low polarity such as the hydrophobic interior of a protein or in a lipid membrane environment and Trp fluorescence in proteins has been classified into five classes by Reshetnyak et al. [34]. According to this classification, the dominant fluorescence around 329 nm corresponds to a class of Trp side chains that are in a relatively non-polar environment and H-bonded in an 2∶1 exciplex that fluoresces at 331 nm [34]. Inspection of the three-dimensional structure of SoPIP2;1 indicated that the Trps of this protein are positioned close to the surface of the protein (see cartoon i in Figure 5A) [22]. SoPIP2;1 reconstituted into detergent-micelles exhibited a maximum fluorescence at 330 nm indicating that the region of the protein where the Trps are located is positioned at the edge of the detergent micelles thereby facilitating the contacts between Trps and water molecules (see cartoon ii in Figure 5A and experimental results in Figure 5B). The easier accessibility of the water to the Trp environment is due to the loosely packed hydrophobic tails of the detergents interacting with the protein in detergent-micelles. A shift to lower wavelength values was always observed when the protein was reconstituted into lipid membranes (representative spectra showed with the E. coli membranes in Figure 5B). It demonstrates that the lipid membrane environment offers a more extensive hydrophobic surface for interaction with the transmembrane protein (cartoon iii in figure 5A). It is also consistent with the higher temperature stability exhibited by the protein in the membrane systems. Thermal stability of the protein was also monitored by following the Trp fluorescence vs. increasing temperature. The Trp fluorescence was quenched at higher temperatures while the wavelength at which maximum emission took place was not affected significantly (Figure 5B and 6).


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)

Fluorescence intensity at the maximum of the emission spectra as a function of temperature.The protein reconstituted in detergent micelles show one principal transitions in comparison with the protein reconstituted in lipid membranes where at least three different transitions can be observed. Fluorescence intensity values are normalized to the higher intensity value observed with the protein in micelles suspension at 20°C.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0014674-g006: Fluorescence intensity at the maximum of the emission spectra as a function of temperature.The protein reconstituted in detergent micelles show one principal transitions in comparison with the protein reconstituted in lipid membranes where at least three different transitions can be observed. Fluorescence intensity values are normalized to the higher intensity value observed with the protein in micelles suspension at 20°C.
Mentions: SoPIP2;1 contains six tryptophan (Trp) residues in the primary sequence. These residues can be used as intrinsic fluorophores for analyzing the protein ternary structure (see the cartoon in Figure 5A) as the Trp fluorescence emission spectrum is sensitive to both the polarity and the dynamics of the environment surrounding the aromatic side chain. Therefore, the variation in the Trp fluorescence emission spectra reports changes in tertiary structure of the protein. Thus the Trp is generally blue-shifted from 350 nm in environments of low polarity such as the hydrophobic interior of a protein or in a lipid membrane environment and Trp fluorescence in proteins has been classified into five classes by Reshetnyak et al. [34]. According to this classification, the dominant fluorescence around 329 nm corresponds to a class of Trp side chains that are in a relatively non-polar environment and H-bonded in an 2∶1 exciplex that fluoresces at 331 nm [34]. Inspection of the three-dimensional structure of SoPIP2;1 indicated that the Trps of this protein are positioned close to the surface of the protein (see cartoon i in Figure 5A) [22]. SoPIP2;1 reconstituted into detergent-micelles exhibited a maximum fluorescence at 330 nm indicating that the region of the protein where the Trps are located is positioned at the edge of the detergent micelles thereby facilitating the contacts between Trps and water molecules (see cartoon ii in Figure 5A and experimental results in Figure 5B). The easier accessibility of the water to the Trp environment is due to the loosely packed hydrophobic tails of the detergents interacting with the protein in detergent-micelles. A shift to lower wavelength values was always observed when the protein was reconstituted into lipid membranes (representative spectra showed with the E. coli membranes in Figure 5B). It demonstrates that the lipid membrane environment offers a more extensive hydrophobic surface for interaction with the transmembrane protein (cartoon iii in figure 5A). It is also consistent with the higher temperature stability exhibited by the protein in the membrane systems. Thermal stability of the protein was also monitored by following the Trp fluorescence vs. increasing temperature. The Trp fluorescence was quenched at higher temperatures while the wavelength at which maximum emission took place was not affected significantly (Figure 5B and 6).

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