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Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.

Naturally occurring photosynthetic systems use elaborate pathways of self-repair to limit the impact of photo-damage. Here, we demonstrate a complex consisting of two recombinant proteins, phospholipids and a carbon nanotube that mimics this process. The components self-assemble into a configuration in which an array of lipid bilayers aggregate on the surface of the carbon nanotube, creating a platform for the attachment of light-converting proteins. The system can disassemble upon the addition of a surfactant and reassemble upon its removal over an indefinite number of cycles. The assembly is thermodynamically metastable and can only transition reversibly if the rate of surfactant removal exceeds a threshold value. Only in the assembled state do the complexes exhibit photoelectrochemical activity. We demonstrate a regeneration cycle that uses surfactant to switch between assembled and disassembled states, resulting in an increased photoconversion efficiency of more than 300% over 168 hours and an indefinite extension of the system lifetime. [ABSTRACT FROM AUTHOR]

One of the biggest challenges in pharmaceutical research is obtaining integral membrane proteins in a functional, solubilized, and monodisperse state that provides a native-like environment that maintains the spectrum of in vivo activities. Many of these integral membrane proteins are receptors, enzymes, or other macromolecular assemblies that are important drug targets. An example is the general class of proteins composed of seven-transmembrane segments (7-TM) as exemplified by the G-protein-coupled receptors. In this article, we describe a simple system for self-assembling bacteriorhodopsin, as a model protein containing 7-TM helices, with phospholipids to form a nanometer-scale soluble bilayer structure encircled by a 200 amino acid scaffold protein. The result is the single molecule incorporation of an integral membrane protein target into a soluble and monodisperse structure that allows the structural and functional tools of solution biochemistry to be applied. [ABSTRACT FROM AUTHOR]

Integral membrane G protein-coupled receptors (GPCRs) compose the single most prolific class of drug targets, yet significant functional and structural questions remain unanswered for this superfamily. A primary reason for this gap in understanding arises from the difficulty of forming soluble, monodisperse receptor membrane preparations that maintain the transmembrane signaling activity of the receptor and provide robust biophysical and biochemical assay systems. Here we report a technique for self-assembling functional beta2-adrenergic receptor (beta2AR) into a nanoscale phospholipid bilayer system (Nanodisc) that is highly soluble in aqueous solution. The approximately 10-nm nanobilayer particles contain beta2AR in a native-like phospholipid bilayer domain of approximately 100 phospholipid molecules circumferentially bound by a membrane scaffold protein (MSP). The resulting construct allows for access to the physiologically intracellular and extracellular faces of the receptor and thus allows unrestricted access of antagonists, agonists, and G proteins. These Nanodisc-solubilized GPCRs can be directly purified by normal chromatographic procedures. We define the resultant Nanodisc-embedded monomeric beta2AR by antagonist and agonist binding isotherms and demonstrate faithful G protein coupling.

One of the biggest challenges in pharmaceutical research is obtaining integral membrane proteins in a functional, solubilized, and monodisperse state that provides a native-like environment that maintains the spectrum of in vivo activities. Many of these integral membrane proteins are receptors, enzymes, or other macromolecular assemblies that are important drug targets. An example is the general class of proteins composed of seven-transmembrane segments (7-TM) as exemplified by the G-protein-coupled receptors. In this article, we describe a simple system for self-assembling bacteriorhodopsin, as a model protein containing 7-TM helices, with phospholipids to form a nanometer-scale soluble bilayer structure encircled by a 200 amino acid scaffold protein. The result is the single molecule incorporation of an integral membrane protein target into a soluble and monodisperse structure that allows the structural and functional tools of solution biochemistry to be applied.