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-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.

Figure 1. Far-UV CD spectra of SoPIP2;1.

The measurements were obtained at 20°C in phosphate buffer, pH 7.5, NaCl 150 mM containing 1% OG and in different lipid membranes.

Figure 2. Thermal unfolding of the secondary structure of SoPIP2;1.

Protein was reconstituted in A) OG micelles, B) E. coli lipid membranes, C) POPE∶POPC∶POPS∶Ergosterol membranes and D) POPE∶POPC membranes. Temperatures are represented by the grayscale colors from black, 20°C to very light gray, 95°C.

Figure 3. 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 (◊).

Figure 4. Thermal denaturation of SoPIP2;1.

It can be visualized by SDS-PAGE since large aggregates are formed that cannot enter the gel. After incubation at room temperature the normal bands corresponding to monomeric, dimeric, trimeric and tetrameric SoPIP2;1 are evident. No aggregates were found at 50°C whereas at the melting point, 58°C, as determined by CD, part of the protein was aggregated. At 65°C the aggregation was complete. The samples were incubated at the indicated temperatures (25–65°C) for 10 minutes before sample loading buffer was added followed by 10 minutes incubation at room temperature. St, standard with indicated Mw in kDa in the left side of the gel.

Figure 5. Fluorescence spectroscopy analysis of the protein ternary structure behavior.

A) Cartoons illustrating the Trp residues (green) position in the protein ternary structure SoPIP2;1. The structure derives from the PDB file 1Z98 using VMD software is shown in i , an illustration of the protein position in detergent micelles in ii and in lipid bilayers in iii. B) Thermal unfolding of SoPIP2;1 monitored by tryptophan fluorescence. To the left, the protein is reconstituted into OG micelles and to the right the protein is reconstituted into E. coli lipid membranes. Temperatures are represented by the grayscale colors from black, 20°C to very light gray, 95°C.

Figure 6. 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.