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

Abstract

BackgroundSoPIP2;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 FindingWe 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/SignificanceThe 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.

Aquaporin 4 molecular mimicry and implications for neuromyelitis optica

“…Asians are major consumers of both soybean and spinach (Lucier, et al, 2004; Schryver, et al, 2007). Plant aquaporins, are highly stable (Plasencia, et al, 2011), further supporting the hypothesis that they may survive harsh food preparation conditions and reach the gastrointestinal (GI) system of humans as intact proteins/peptides where they may be antigenic. In fact human AQP4 itself is a resident water channel in the stomach (Laforenza, 2012).…”

“…Five plant aquaporin families, namely PIP, TIP, NIP, SIP and XIP, have been well studied and characterized structurally and functionally (Baiges, et al, 2002; Johnson, et al, 1990; Maurel, 2007). Spinach leaf plasma membranes express two thermally stable members of the aquaporin family, PM28A (SoPIP-2) and PM28C (SoPIP-1), which together constitute 20% of the integral membrane proteins (Johansson, et al, 1998; Kukulski, et al, 2005; Plasencia, et al, 2011). Soybean aquaporins occur in the germinating seeds in addition to root nodules (Dean, et al, 1999; Fleurat-Lessard, et al, 2005; Rivers, et al, 1997).…”

Aquaporin–graphene interface: relevance to point-of-care device for renal cell carcinoma and desalination

“…Plasencia et al [5] rotamer of the H193 side-chain (in white) may be adopted which is within hydrogen bonding distance of D28.…”

Aquaporin Biomimetic Membranes

“…Alternatively, the process of optimizing the interaction between membrane, protein (c.f. [64][65][66]), and yield may directly affect how much of functional protein content can be integrated into the final product (c.f. [67]).…”