The directed cooperative assembly of proteorhodopsin into 2D and 3D polarized arrays

Abstract

Proteorhodopsin is the membrane protein used by marine bacterioplankton as a light-driven proton pump. Here, we describe a rapid cooperative assembly process directed by universal electrostatic interactions that spontaneously organizes proteorhodopsin molecules into ordered arrays with well defined orientation and packing density. We demonstrate the charge density-matching mechanism that selectively controls the assembly process. The interactions among different components in the system are tuned by varying their charge densities to yield different organized transmembrane protein arrays: (i) a bacteriorhodopsin purple membrane-like structure where proteorhodopsin molecules are cooperatively arranged with charged lipids into a 2D hexagonal lattice; (ii) selected liquid-crystalline states in which crystalline lamellae made up of the coassembled proteorhodopsin and charged lipid molecules are coupled three-dimensionally with polarized proteorhodopsin orientation persisting through the macroscopic scale. Understanding this rapid electrostatically driven assembly process sheds light on organizing membrane proteins in general, which is a prerequisite for membrane protein structural and mechanistic studies as well as in vitro applications.cationic lipid-proteorhodopsin complexes ͉ membrane protein arrays ͉ membrane protein crystallization ͉ light-driven proton pump ͉ photovoltaics M embrane proteins (MPs) play an essential role in determining the interactions of biological systems with their surroundings. As gatekeepers of cellular boundaries, they are involved with matter, energy, and information transport to maintain life activities, and also with drug delivery to cure diseases. MPs are estimated to represent 20-30% of the currently sequenced genomes (1), and are targets for Ϸ70% of all drugs in the market (2). Although the sequence and function of many MPs have become known, their structures have only been solved at a rate of Ϸ0.2% of that of soluble proteins (3). Little is known about how to harness their unique biological functions in practical devices.Long-range ordering is the prerequisite for structure determination, and unidirectional packing is needed for many in vitro applications depending on MP orientation because the activities of most MPs have vectorial aspects (4). The key problem that hinders MP assembly is their amphiphilic character. Except for a very limited number of MPs that form ordered crystals naturally, most MPs require detergents to free them from their native membranes. Although detergent-solubilized MPs with the surrounding detergent micelle in place could be crystallized (5), the orientation of MPs in those crystals is more or less isotropic, and the effect of lacking the biological membrane confinement on MP structure and mechanistic properties is always a concern. Detergent-assisted reconstitution is the most commonly used strategy to form 2D proteolipsome arrays (6). The MP order in those arrays depends critically on the detergent removal kinetics. Moreover, the lipid versus protein rat...

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

“…Interestingly, insertion into the lipid bilayer is required for pR to be functional. The structure of proteorhodopsin also gives it an inherent asymmetric charge density (24); the C-terminal has an overall positive charge because of an abundance of positively charged residues, and the N-terminal contains a number of negatively charged residues. To investigate whether this charge asymmetry could produce preferential orientation during insertion into the bilayer, we compared proteoliposomes created by pR insertion into cationic, anionic, and charge-neutral liposomes ( Fig.…”

“…A previous study by Liang et al (24) has calculated pH-dependent effective charges associated with pR extramembrane domains at different pH levels. Those calculations show that at pH 5, the N-terminal has an effective FIGURE 2 Limited proteolysis with proteinase K (ProtK) shows that pR orientation in proteoliposomes depends on the lipid composition.…”

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