RESUMO
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 â¼16 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.
Assuntos
Luz , Nanoestruturas/química , Conformação Proteica/efeitos da radiação , Rodopsina/química , Transducina/química , Animais , Bovinos , Estrutura Secundária de Proteína , Rodopsina/metabolismo , Rodopsina/efeitos da radiação , Marcadores de Spin , Transducina/metabolismo , Transducina/efeitos da radiaçãoRESUMO
We have designed and implemented a practical nanoelectronic interface to G-protein coupled receptors (GPCRs), a large family of membrane proteins whose roles in the detection of molecules outside eukaryotic cells make them important pharmaceutical targets. Specifically, we have coupled olfactory receptor proteins (ORs) with carbon nanotube transistors. The resulting devices transduce signals associated with odorant binding to ORs in the gas phase under ambient conditions and show responses that are in excellent agreement with results from established assays for OR-ligand binding. The work represents significant progress on a path toward a bioelectronic nose that can be directly compared to biological olfactory systems as well as a general method for the study of GPCR function in multiple domains using electronic readout.
Assuntos
Biomimética/instrumentação , Técnicas Biossensoriais/instrumentação , Equipamentos e Provisões Elétricas , Nanotecnologia/instrumentação , Receptores Odorantes/metabolismo , Animais , Células HEK293 , Humanos , Camundongos , Nanotubos de Carbono/química , Transistores EletrônicosRESUMO
G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.
Assuntos
Arrestinas/metabolismo , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Rodopsina/metabolismo , Transdução de Sinais/fisiologia , Visão Ocular/fisiologia , Sequência de Aminoácidos , Animais , Arrestinas/genética , Bovinos , Eletroquímica , Fluorescência , Leucina/metabolismo , Leucina/farmacologia , Lipídeos/química , Dados de Sequência Molecular , Mutação , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Rodopsina/química , Rodopsina/genética , Trítio , beta-ArrestinasRESUMO
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.
Assuntos
Eletroquímica , Fotoquímica , Energia Solar , Bicamadas Lipídicas , Nanotubos de Carbono , Fosfolipídeos/química , Proteínas Recombinantes/químicaRESUMO
Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane proteins for biophysical, enzymatic or structural investigations. This means for rendering membrane proteins soluble at the single molecule level offers advantages over liposomes or detergent micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structure and ready access to both sides of the phospholipid bilayer domain. Thus the Nanodisc system provides a novel platform for understanding membrane protein function. We provide an overview of the Nanodisc approach and document through several examples many of the applications to the study of the structure and function of integral membrane proteins.
Assuntos
Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Nanoestruturas , Multimerização Proteica/fisiologia , Animais , Humanos , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Modelos Biológicos , Modelos Moleculares , Nanoestruturas/química , Propriedades de SuperfícieRESUMO
Nanodiscs are nanometer scale planar membranes of controlled size that are rendered soluble in aqueous solution via an encircling amphipathic membrane scaffold protein "belt" (Bayburt, T. H., Grinkova, Y. V., and Sligar, S. G. (2002) Nano. Lett. 2, 853-856). Integral membrane proteins can be self-assembled into the Nanodisc bilayer with defined stoichiometry, which allows an unprecedented opportunity to investigate the nature of the oligomerization state of a G-protein-coupled receptor and its coupling to heterotrimeric G-proteins. We generated Nanodiscs having one and two rhodopsins present in the 10-nm-diameter lipid bilayer domain. Efficient transducin activation and isolation of a high affinity transducin-metarhodopsin II complex was demonstrated for a monodisperse and monomeric receptor. A population of Nanodiscs containing two rhodopsins was generated using an increased ratio of receptor to membrane scaffold protein in the self-assembly mixture. The two-rhodopsin population was isolated and purified by density gradient centrifugation. Interestingly, in this case, only one of the two receptors present in the Nanodisc was able to form a stable metarhodopsin II-G-protein complex. Thus there is clear evidence that a monomeric rhodopsin is capable of full coupling to transducin. Importantly, presumably due to steric interactions, it appears that only a single receptor in the Nanodiscs containing two rhodopsins can interact with G-protein. These results have important implications for the stoichiometry of receptor-G-protein coupling and cross talk in signaling pathways.
Assuntos
Bicamadas Lipídicas/química , Complexos Multiproteicos/química , Nanopartículas/química , Rodopsina/química , Transducina/química , Animais , Humanos , Complexos Multiproteicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rodopsina/genética , Transducina/genéticaRESUMO
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.
Assuntos
Bicamadas Lipídicas/metabolismo , Nanoestruturas/química , Nanotecnologia , Receptores Adrenérgicos beta 2/isolamento & purificação , Receptores Adrenérgicos beta 2/metabolismo , Linhagem Celular , Cromatografia em Gel , Detergentes/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Bicamadas Lipídicas/química , Modelos Biológicos , Tamanho da Partícula , Fosfatidilcolinas/química , Ligação Proteica , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Água/químicaRESUMO
Nanodiscs, phospholipid bilayer assemblies of controlled size, were used to self-assemble bacteriorhodopsin (bR) into single trimers. Self-assembly at optimal bR to Nanodisc and phospholipid stoichiometry yielded particles containing three bR molecules. Analysis of solution small angle X-ray scattering indicated that bacteriorhodopsin is embedded in a discoidal phospholipid bilayer structure. Formation of trimers, as evidenced by visible circular dichroism of the retinal absorbance bands, is facilitated in Nanodiscs at a specific size threshold, suggesting that a critical bilayer area or amount of lipid is necessary to maintain a native oligomeric state. The lipid to bR ratio in the assembly process was also found to be an important factor in determining oligomerization state. These nanoscale bilayers offer the opportunity to understand and control the assembly of oligomeric integral membrane proteins critical to macromolecular recognition and cellular signaling.
Assuntos
Bacteriorodopsinas/química , Bicamadas Lipídicas/química , Colatos/química , Dicroísmo Circular , Dimiristoilfosfatidilcolina/química , Halobacterium salinarum , Nanoestruturas , Polímeros , Espalhamento de Radiação , Raios XRESUMO
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.
Assuntos
Bacteriorodopsinas/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanotubos/química , Sequência de Aminoácidos , Halobacterium salinarum/química , Bicamadas Lipídicas/síntese química , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Fosfolipídeos/química , Ligação ProteicaRESUMO
One of the biggest challenges in the field of proteomics is obtaining functional membrane proteins solubilized and dispersed into a physiologically relevant environment that maintains the spectrum of in vivo activities. Here we describe a system composed of nanoscale self-assembled particles, termed Nanodiscs, which contain a single phospholipid bilayer stabilized by an encircling membrane scaffold protein (MSP). Using microsomal membranes of baculovirus-infected Spodoptera frugiperda (Sf9) insect cells overexpressing an N-terminally anchored cytochrome P450 monoxygenase (P450), we demonstrate that target membrane proteins can be directly solubilized and incorporated into distinct populations of Nanodiscs, which can be separated by size chromatography. We show that formation of these Nanodiscs from insect cell membranes allows for the compartmentalization into soluble nanostructures that provide a natural membrane bilayer that avoids the aggregation of membrane proteins often encountered in other reconstitution procedures. Lipid composition analysis and substrate binding analysis of size-fractionated Nanodiscs arrayed in microtiter plates further demonstrates that the Nanodisc system effectively disperses the overexpressed membrane protein into monodispersed bilayers containing biochemically defined lipid components and the target protein in its native from suitable for sensitive high-throughput substrate binding analysis.
Assuntos
Materiais Biomiméticos/química , Sistema Enzimático do Citocromo P-450/química , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Nanotubos/química , Spodoptera/enzimologia , Animais , Materiais Biomiméticos/síntese química , Sistema Enzimático do Citocromo P-450/metabolismo , Bicamadas Lipídicas/síntese química , Substâncias Macromoleculares , Membranas Artificiais , Nanotecnologia/métodos , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidade , Spodoptera/química , Spodoptera/genéticaRESUMO
The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.