RESUMO
BACKGROUND: The reconstitution of membrane proteins and complexes into nanoscale lipid bilayer structures has contributed significantly to biochemical and biophysical analyses. Current methods for performing such reconstitutions entail an initial detergent-mediated step to solubilize and isolate membrane proteins. Exposure to detergents, however, can destabilize many membrane proteins and result in a loss of function. Amphipathic copolymers have recently been used to stabilize membrane proteins and complexes following suitable detergent extraction. However, the ability of these copolymers to extract proteins directly from native lipid bilayers for subsequent reconstitution and characterization has not been explored. RESULTS: The styrene-maleic acid (SMA) copolymer effectively solubilized membranes of isolated mitochondria and extracted protein complexes. Membrane complexes were reconstituted into polymer-bound nanoscale discs along with endogenous lipids. Using respiratory Complex IV as a model, these particles were shown to maintain the enzymatic activity of multicomponent electron transporting complexes. CONCLUSIONS: We report a novel process for reconstituting fully operational protein complexes directly from cellular membranes into nanoscale lipid bilayers using the SMA copolymer. This facile, single-step strategy obviates the requirement for detergents and yields membrane complexes suitable for structural and functional studies.
Assuntos
Biotecnologia/métodos , Proteínas de Membrana/isolamento & purificação , Membranas Mitocondriais/enzimologia , Complexos Multienzimáticos/isolamento & purificação , Nanopartículas/química , Maleatos/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Nanopartículas/metabolismo , Poliestirenos/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl-ß-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl-ß-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature (T(m)) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the T(m) as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation (E(act)) was calculated from the scan rate dependence of the T(m) and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The E(act) as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the E(act) to rhodopsin denaturation.
Assuntos
Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Absorção/efeitos dos fármacos , Animais , Varredura Diferencial de Calorimetria , Bovinos , Membrana Celular/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Glucosídeos/farmacologia , Cinética , Osmose/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Solubilidade/efeitos dos fármacos , Fatores de Tempo , Temperatura de Transição/efeitos dos fármacosRESUMO
The outer segment of rod photoreceptor cells is responsible for initiating visual signal transduction when light levels are low. It consists of stacked disk membranes surrounded by the plasma membrane and is under continuous renewal. Disk membranes are synthesized at the base of the outer segment and are progressively displaced toward the apical tip where they are phagocytosed by the pigment epithelium. This process takes approximately ten days during which time the lipid composition of the disk membrane is modified. Disk membranes become progressively lower in cholesterol and higher in unsaturated phospholipids during spatial displacement. The modification of disk composition is likely important for normal function of the rod cell. Flow cytometry is shown in this study to be a powerful tool to assess differences among rod outer segment disk membranes. First, the feasibility of flow cytometry to detect individual osmotically intact disks was established. Fluorescent beads of 1.0 µm, 0.5 µm, 0.2 µm, and 0.1 µm diameter were used to demonstrate that side-scatter intensity measured on a log scale corresponds to bead diameter. The intensity of disk side scatter predominantly corresponded to 0.1-0.2 µm diameter beads. We next applied this technique to detect caveolin-1, peripherin/rds, and GM1 in disk membranes. Caveolin-1 was detected with FITC-labeled anti-caveolin-1, peripherin/rds with Cy3 labeled anti-peripherin/rds, and GM1 with FITC-labeled cholera toxin subunit B. Fluorescence due to caveolin-1 and peripherin/rds binding was detected in 80% and 70% respectively of the events that corresponded to scattering attributed to disks. Fluorescence attributed to GM1 was detected in 60% of the events that corresponded to disk scattering. The intensity of fluorescence due to caveolin-1 and peripherin/rds labeling was directly proportional to the intensity of side scatter, indicating that both caveolin-1 and peripherin/rds concentrations are uniform among all the disks. Fluorescence intensity of GM1 labeled disks was independent of side-scatter intensity indicating GM1 is heterogeneously distributed among the disks. These results are consistent with integral membrane proteins such as caveolin-1, and peripherin/rds remaining in the disk bilayer as the disks are apically displaced. The GM1 distribution may be similar to the spatial distribution of cholesterol in the outer segment.
Assuntos
Caveolina 1/metabolismo , Membrana Celular/metabolismo , Gangliosídeo G(M1)/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Animais , Bovinos , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Microesferas , PeriferinasRESUMO
The photochemical and thermal stability of the detergent-solubilized blue- and green-absorbing proteorhodpsins, BPR and GPR, respectively, are investigated to determine the viability of these proteins for photonic device applications. Photochemical stability is studied by using pulsed laser excitation and differential UV-vis spectroscopy to assign the photocyclicity. GPR, with a cyclicity of 7 × 10(4) photocycles protein(-1), is 4-5 times more stable than BPR (9 × 10(3) photocycles protein(-1)), but is less stable than native bacteriorhodopsin (9 × 10(5) photocycles protein(-1)) or the 4-keto-bacteriorhodopsin analogue (1 × 10(5) photocycles protein(-1)). The thermal stabilities are assigned by using differential scanning calorimetry and thermal bleaching experiments. Both proteorhodopsins display excellent thermal stability, with melting temperatures above 85 °C, and remain photochemically stable up to 75 °C. The biological relevance of our results is also discussed. The lower cyclicity of BPR is found to be adequate for the long-term biological function of the host organism at ocean depths of 50 m or more.
Assuntos
Biotecnologia/métodos , Equipamentos e Provisões Elétricas , Processos Fotoquímicos , Rodopsina/química , Temperatura , Detergentes/química , Escherichia coli/genética , Glucosídeos/química , Estabilidade Proteica , Rodopsina/genética , Rodopsinas Microbianas , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
Structural data on membrane proteins, while crucial to understanding cellular function, are scarce due to difficulties in applying to membrane proteins the common techniques of structural biology. Fragments of membrane proteins have been shown to reflect, in many cases, the secondary structure of the parent protein with fidelity and are more amenable to study. This chapter provides many examples of how the study of membrane protein fragments has provided new insight into the structure of the parent membrane protein.
Assuntos
Proteínas de Membrana/química , Espectroscopia de Ressonância Magnética , Modelos MolecularesRESUMO
The photoreceptor rhodopsin is a G-protein coupled receptor that has recently been proposed to exist as a dimer or higher order oligomer, in contrast to the previously described monomer, in retinal rod outer segment disk membranes. Rhodopsin exhibits considerably greater thermal stability than opsin (the bleached form of the receptor), which is reflected in an approximately 15 degrees C difference in the thermal denaturation temperatures (T(m)) of rhodopsin and opsin as measured by differential scanning calorimetry. Here we use differential scanning calorimetry to investigate the effect of partial bleaching of disk membranes on the T(m) of rhodopsin and of opsin in native disk membranes, as well as in cross-linked disk membranes in which rhodopsin dimers are known to be present. The T(m)s of rhodopsin and opsin are expected to be perturbed if mixed oligomers are present. The T(m) remained constant for rhodopsin and opsin in native disks regardless of the level of bleaching. In contrast, the T(m) of cross-linked rhodopsin in disk membranes was dependent on the extent of bleaching. The energy of activation for denaturation of rhodopsin and cross-linked rhodopsin was calculated. Cross-linking rhodopsin significantly decreased the energy of activation. We conclude that in native disk membranes, rhodopsin behaves predominantly as a monomer.
Assuntos
Membrana Celular/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/citologia , Opsinas de Bastonetes/metabolismo , Animais , Varredura Diferencial de Calorimetria , Bovinos , Reagentes de Ligações Cruzadas/metabolismo , Dimerização , Cinética , Desnaturação Proteica , Estrutura Terciária de Proteína , Rodopsina/química , Opsinas de Bastonetes/química , Temperatura de TransiçãoRESUMO
Because of their central role in regulation of cellular function, structure/function relationships for G-protein coupled receptors (GPCR) are of vital importance, yet only recently have sufficient data been obtained to begin mapping those relationships. GPCRs regulate a wide range of cellular processes, including the senses of taste, smell, and vision, and control a myriad of intracellular signaling systems in response to external stimuli. Many diseases are linked to GPCRs. A critical need exists for structural information to inform studies on mechanism of receptor action and regulation. X-ray crystal structures of only one GPCR, in an inactive state, have been obtained to date. However considerable structural information for a variety of GPCRs has been obtained using non-crystallographic approaches. This review begins with a review of the very earliest GPCR structural information, mostly derived from rhodopsin. Because of the difficulty in crystallizing GPCRs for X-ray crystallography, the extensive published work utilizing alternative approaches to GPCR structure is reviewed, including determination of three-dimensional structure from sparse constraints. The available X-ray crystallographic analyses on bovine rhodopsin are reviewed as the only available high-resolution structures for any GPCR. Structural information available on ligand binding to several receptors is included. The limited information on excited states of receptors is also reviewed. It is concluded that while considerable basic structural information has been obtained, more data are needed to describe the molecular mechanism of activation of a GPCR.
Assuntos
Receptores Acoplados a Proteínas G/química , Cristalografia por Raios X , Humanos , Conformação ProteicaRESUMO
The photoreceptor rod outer segment (ROS) provides a unique system in which to investigate the role of cholesterol, an essential membrane constituent of most animal cells. The ROS is responsible for the initial events of vision at low light levels. It consists of a stack of disk membranes surrounded by the plasma membrane. Light capture occurs in the outer segment disk membranes that contain the photopigment, rhodopsin. These membranes originate from evaginations of the plasma membrane at the base of the outer segment. The new disks separate from the plasma membrane and progressively move up the length of the ROS over the course of several days. Thus the role of cholesterol can be evaluated in two distinct membranes. Furthermore, because the disk membranes vary in age it can also be investigated in a membrane as a function of the membrane age. The plasma membrane is enriched in cholesterol and in saturated fatty acids species relative to the disk membrane. The newly formed disk membranes have 6-fold more cholesterol than disks at the apical tip of the ROS. The partitioning of cholesterol out of disk membranes as they age and are apically displaced is consistent with the high PE content of disk membranes relative to the plasma membrane. The cholesterol composition of membranes has profound consequences on the major protein, rhodopsin. Biophysical studies in both model membranes and in native membranes have demonstrated that cholesterol can modulate the activity of rhodopsin by altering the membrane hydrocarbon environment. These studies suggest that mature disk membranes initiate the visual signal cascade more effectively than the newly synthesized, high cholesterol basal disks. Although rhodopsin is also the major protein of the plasma membrane, the high membrane cholesterol content inhibits rhodopsin participation in the visual transduction cascade. In addition to its effect on the hydrocarbon region, cholesterol may interact directly with rhodopsin. While high cholesterol inhibits rhodopsin activation, it also stabilizes the protein to denaturation. Therefore the disk membrane must perform a balancing act providing sufficient cholesterol to confer stability but without making the membrane too restrictive to receptor activation. Within a given disk membrane, it is likely that cholesterol exhibits an asymmetric distribution between the inner and outer bilayer leaflets. Furthermore, there is some evidence of cholesterol microdomains in the disk membranes. The availability of the disk protein, rom-1 may be sensitive to membrane cholesterol. The effects exerted by cholesterol on rhodopsin function have far-reaching implications for the study of G-protein coupled receptors as a whole. These studies show that the function of a membrane receptor can be modulated by modification of the lipid bilayer, particularly cholesterol. This provides a powerful means of fine-tuning the activity of a membrane protein without resorting to turnover of the protein or protein modification.
Assuntos
Colesterol/metabolismo , Segmento Externo da Célula Bastonete/fisiologia , Animais , Membrana Celular/metabolismo , Colesterol/biossíntese , Colesterol/farmacocinética , Humanos , Hidrocarbonetos/metabolismo , Modelos Biológicos , Disco Óptico/metabolismo , Disco Óptico/fisiologia , Fosfolipídeos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Doenças Retinianas/metabolismo , Doenças Retinianas/fisiopatologia , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/anatomia & histologia , Segmento Externo da Célula Bastonete/metabolismo , Visão Ocular/fisiologiaRESUMO
Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds. This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding. In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds. Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes. To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed. A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site. Protein expression was induced in E. coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column. Purity was confirmed by SDS-PAGE and Western blot analysis. The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R. Molday). The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing. Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps. A common feature of most membrane fusion proteins is a change in conformation upon membrane association. Structural changes in the C-terminal polypeptide were investigated using far UV CD. The far UV CD spectra of the purified C-terminal polypeptide indicated substantial alpha-helical content in the wt peptide in isotonic aqueous buffer. An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in alpha-helical content of the polypeptide. These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in alpha-helical content.
Assuntos
Proteínas do Olho/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Fusão de Membrana/fisiologia , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Western Blotting/métodos , Dicroísmo Circular/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Produtos do Gene nef/metabolismo , Glutationa Transferase/metabolismo , Humanos , Metabolismo dos Lipídeos , Mutação , Periferinas , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , SolubilidadeRESUMO
G protein-coupled receptors (GPCRs) are a family of seven transmembrane helical proteins that initiate a cellular response to an environmental signal. Once activated by an extracellular signal, GPCRs trigger the intracellular signal transduction cascade by activating a heterotrimeric G protein. The interaction between the G protein and the receptor, which triggers the signal transduction, is the focus of intense interest. Three-dimensional structures of the ground state of only one GPCR, rhodopsin, are currently available, but since the G protein cannot bind to this structure, these structures did not lead to an understanding of the activation process. The recent publication of an excited state structure for the same GPCR (and comparison to the ground state structures), in conjunction with other recent biochemical data, provides new insight into G protein activation. We find that the structure data and the biochemical data, for the first time, point to a specific mode of interaction between the G protein and the receptor. Furthermore, we find that transducin (G(t)) must alter its conformation to bind to the activated receptor; the "lock and key" fit heretofore expected is likely not the correct model. We suggest that a conformational distortion, driven by the energy of binding, is induced in G(t) when it binds to the activated receptor. The conformational change in turn enables the exchange of GTP for GDP and the dissociation of the subunits. This is an example of "induced fit" originally proposed by Koshland to describe enzyme-substrate interactions.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/química , Receptores de Superfície Celular/química , Rodopsina/análogos & derivados , Rodopsina/química , Animais , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Conformação Proteica , Receptores de Superfície Celular/fisiologia , Rodopsina/metabolismoRESUMO
Bovine rhodopsin is the prototypical G protein coupled receptor (GPCR). It was the first GPCR to be obtained in quantity and studied in detail. It is also the first GPCR for which detailed three dimensional structural information has been obtained. Reviewed here are the experiments leading up to the high resolution structure determination of rhodopsin and the most recent structural information on the activation and stability of this integral membrane protein.
Assuntos
Proteínas de Membrana/química , Rodopsina/análogos & derivados , Rodopsina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Citoplasma/química , Adaptação à Escuridão , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Estrutura Molecular , Retinaldeído/química , Rodopsina/isolamento & purificação , Rodopsina/fisiologiaRESUMO
The structural changes that accompany activation of a G-protein coupled receptor (GPCR) are not well understood. To better understand the activation of rhodopsin, the GPCR responsible for visual transduction, we report studies on the three-dimensional structure for the activated state of this receptor, metarhodopsin II. Differences between the three-dimensional structure of ground state rhodopsin and metarhodopsin II, particularly in the cytoplasmic face of the receptor, suggest how the receptor is activated to couple with transducin. In particular, activation opens a groove on the surface of the receptor that could bind the N-terminal helix of the G protein, transducin alpha.