RESUMEN
Cannabinoid drugs differ in their rank order of potency to produce analgesia versus other central nervous system effects. We propose that these differences are due to unique agonist-bound cannabinoid CB1 receptor conformations that exhibit different affinities for individual subsets of intracellular signal transduction pathways. In order to test this hypothesis, we have used plasmon-waveguide resonance (PWR) spectroscopy, a sensitive method that can provide direct information about ligand-protein and protein-protein interactions, and can detect conformational changes in lipid-embedded proteins. A recombinant epitope-tagged human cannabinoid CB1 receptor was expressed in insect Sf9 cells, solubilized and purified using two-step affinity chromatography. The purified receptor was incorporated into a lipid bilayer on the surface of the PWR resonator. PWR spectroscopy demonstrated that cannabinoid agonists exhibit high affinity (KD=0.2+/-0.03 nM and 2+/-0.4 nM for CP 55,940 and WIN 55,212-2, respectively) for the purified epitope tagged hCB(1) receptor. Interestingly however, these structurally different cannabinoid agonists shifted the PWR spectra in opposite directions, indicating that CP 55,940 and WIN 55,212-2 binding leads to different hCB1 receptor conformations. Furthermore, PWR experiments also indicated that these CP 55,940-and WIN 55,212-bound hCB1 receptor conformations exhibit slightly different affinities to an inhibitory G protein heterotrimer, Gi1 (KD=27+/-8 nM and KD=10.7+/-4.7 nM, respectively), whereas they strikingly differ in their ability to activate this G protein type.
Asunto(s)
Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/química , Transducción de Señal , Animales , Benzoxazinas/metabolismo , Células Cultivadas , Ciclohexanoles/metabolismo , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Morfolinas/metabolismo , Naftalenos/metabolismo , Conformación Proteica , Receptor Cannabinoide CB1/fisiología , SpodopteraRESUMEN
Plasmon-waveguide resonance (PWR) spectroscopy is a high-sensitivity optical method for characterizing thin films immobilized onto the outer surface of a glass prism coated with thin films of a metal (e.g., silver) and a dielectric (e.g., silica). Resonance excitation by a polarized continuous wave (CW) laser above the critical angle for total internal reflection generates plasmon and waveguide modes, whose evanescent electromagnetic fields are localized on the outer surface and interact with the immobilized sample (in the present case a proteolipid bilayer). Plots of reflected light intensity vs the incident angle of the exciting light constitute a PWR spectrum, whose properties are determined by the refractive index (n), the thickness (t), and the optical extinction at the exciting wavelength (k) of the sample. Plasmon excitation can occur using light polarized both perpendicular (p) and parallel (s) to the plane of the resonator surface, allowing characterization of the structural properties of uniaxially oriented proteolipid films deposited on the surface. As will be demonstrated in what follows, PWR spectroscopy provides a powerful tool for directly observing in real-time microdomain formation (rafts) in such bilayers owing to lateral segregation of both lipids and proteins. In favorable cases, protein trafficking can also be monitored. Spectral simulation using Maxwell's equations allows these raft domains to be characterized in terms of their mass densities and thicknesses.
Asunto(s)
Membrana Dobles de Lípidos/química , Proteolípidos/análisis , Proteolípidos/química , Resonancia por Plasmón de Superficie/métodos , Fosfatasa Alcalina/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Colesterol/química , Simulación por Computador , Humanos , Microdominios de Membrana/química , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Óptica y Fotónica , Fosfatidilcolinas/química , Receptores Opioides delta/metabolismo , Esfingomielinas/químicaRESUMEN
We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Colesterol/metabolismo , Membrana Dobles de Lípidos/química , Fragmentos de Péptidos/metabolismo , Péptidos/metabolismo , Esfingomielinas/metabolismo , Zinc/metabolismo , Péptidos beta-Amiloides/química , Colesterol/química , Membrana Dobles de Lípidos/metabolismo , Fragmentos de Péptidos/química , Péptidos/química , Fosfatidilcolinas/química , Unión Proteica , Análisis Espectral/métodos , Esfingomielinas/química , Zinc/químicaRESUMEN
Plasmon-waveguide resonance (PWR) spectroscopy has been used to study the interactions between ligands that correspond to inhibitors, activators or substrates and three integral membrane proteins representing potential drug targets; cyclooxygenases 1 and 2 (COX-1 and -2), integrin alphaVbeta3, and hepatitis C virus RNA polymerase. The proteins were incorporated into an egg phosphatidylcholine bilayer deposited onto the surface of the PWR resonator, and changes in the amplitude and position of the PWR spectra due to mass density increases and conformational transitions have been used to characterize the kinetics and binding affinities corresponding to these interactions. Although the partition of COX-2 into the bilayer was not as efficient as was the case with the other two proteins, sufficient protein could be incorporated to allow ligand binding to be observed. It was also possible to incorporate COX-1 into a lipid bilayer by adding a suspension of microsomal membrane fragments containing this enzyme to a preformed bilayer, and to observe binding of an inhibitory ligand. The interactions between integrin alphaVbeta3 and two ligands with different in vivo efficacies could be distinguished by both spectral measurements and binding kinetics. In the case of the RNA polymerase, the kinetics of PWR spectral changes upon adding a substrate solution to an enzyme-template complex were consistent with those obtained from direct measurements of enzymatic turnover. These experiments demonstrate the utility of PWR spectroscopy to provide novel information regarding drug interactions with membrane proteins in a lipid environment and to distinguish conformational changes induced by binding of various drug molecules.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Hepacivirus/enzimología , Integrina alfaVbeta3/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Resonancia por Plasmón de Superficie/métodos , Ciclooxigenasa 2 , ARN Polimerasas Dirigidas por ADN/análisis , Sistemas de Liberación de Medicamentos/métodos , Integrina alfaVbeta3/análisis , Prostaglandina-Endoperóxido Sintasas/análisisRESUMEN
Plasmon-waveguide resonance (PWR) spectroscopy has been used to examine solid-supported lipid bilayers consisting of dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), sphingomyelin (SM), and phosphatidylcholine/SM binary mixtures. Spectral simulation of the resonance curves demonstrated an increase in bilayer thickness, long-range order, and molecular packing density in going from DOPC to POPC to SM single component bilayers, as expected based on the decreasing level of unsaturation in the fatty acyl chains. DOPC/SM and POPC/SM binary mixtures yielded PWR spectra that can be ascribed to a superposition of two resonances corresponding to microdomains (rafts) consisting of phosphatidylcholine- and SM-rich phases coexisting within a single bilayer. These were formed spontaneously over time as a consequence of lateral phase separation. Each microdomain contained a small proportion (<20%) of the other lipid component, which increased their kinetic and thermodynamic stability. Incorporation of a glycosylphosphatidylinositol-linked protein (placental alkaline phosphatase) occurred within each of the single component bilayers, although the insertion was less efficient into the DOPC bilayer. Incorporation of placental alkaline phosphatase into a DOPC/SM binary bilayer occurred with preferential insertion into the SM-rich phase, although the protein incorporated into both phases at higher concentrations. These results demonstrate the utility of PWR spectroscopy to provide insights into raft formation and protein sorting in model lipid membranes.
Asunto(s)
Membrana Dobles de Lípidos/química , Microdominios de Membrana/química , Fosfatidilcolinas/química , Esfingomielinas/química , Resonancia por Plasmón de SuperficieRESUMEN
Here we report, for the first time, the formation of a biomimetic covalently imprinted polymeric sensor for a target ligand, the delta-opioid G-protein coupled receptor agonist DPDPE, which reproducibly exhibits subpicomolar binding affinity in an aqueous environment. In addition to having a well-defined and homogeneous binding site, the imprinted polymer template is quite stable to storage in both the dry and wet states and has at least 6 orders of magnitude higher affinities than exhibited by similar peptide-based molecular-imprinted polymers (MIPs) thus far. A highly sensitive optical detection methodology, plasmon-waveguide resonance spectroscopy, was employed, capable of measuring binding in real time and discriminating between ligand molecules, without requiring labeling protocols (fluorophores or radioisotopes). The DPDPE-imprinted polymer showed a broad structure-activity relationship profile, not unlike that found for protein receptors. Such sensitivity and robustness of MIPs suggests potential applications ranging from biowarfare agent detection to pharmaceutical screening.
Asunto(s)
Encefalina D-Penicilamina (2,5)/análisis , Receptores Opioides delta/metabolismo , Resonancia por Plasmón de Superficie/métodos , Ligandos , Oligopéptidos/análisis , Oligopéptidos/metabolismo , Polímeros/química , Polímeros/metabolismo , Receptores Opioides delta/agonistas , Receptores Opioides delta/análisis , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , alfa-MSH/análogos & derivados , alfa-MSH/análisis , alfa-MSH/metabolismoRESUMEN
Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that can be used to probe the molecular interactions occurring within anisotropic proteolipid membranes in real time without requiring molecular labeling. This method directly monitors mass density, conformation, and molecular orientation changes occurring in such systems and allows determination of protein-ligand binding constants and binding kinetics. In the present study, PWR has been used to monitor the incorporation of the human beta(2)-adrenergic receptor into a solid-supported egg phosphatidylcholine lipid bilayer and to follow the binding of full agonists (isoproterenol, epinephrine), a partial agonist (dobutamine), an antagonist (alprenolol), and an inverse agonist (ICI-118,551) to the receptor. The combination of differences in binding kinetics and the PWR spectral changes point to the occurrence of multiple conformations that are characteristic of the type of ligand, reflecting differences in the receptor structural states produced by the binding process. These results provide new evidence for the conformational heterogeneity of the liganded states formed by the beta(2)-adrenergic receptor.
Asunto(s)
Receptores Adrenérgicos beta 2/química , Resonancia por Plasmón de Superficie/métodos , Agonistas Adrenérgicos/química , Agonistas de Receptores Adrenérgicos beta 2 , Antagonistas de Receptores Adrenérgicos beta 2 , Antagonistas Adrenérgicos beta/química , Alprenolol/química , Anisotropía , Epinefrina/química , Humanos , Isoproterenol/química , Cinética , Ligandos , Membrana Dobles de Lípidos/química , Fosfatidilcolinas/química , Propanolaminas/química , Unión Proteica , Proteolípidos/química , Análisis Espectral/métodosRESUMEN
We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.
Asunto(s)
Proteínas Bacterianas/metabolismo , Púrpura de Bromocresol/metabolismo , Colorantes/metabolismo , Mutación , Fenolsulfonftaleína/análogos & derivados , Fenolsulfonftaleína/metabolismo , Fotorreceptores Microbianos , Protones , Proteínas Bacterianas/genética , Ectothiorhodospira/metabolismo , CinéticaRESUMEN
Understanding structure-function relationships and mechanisms of signal transduction in G-protein-coupled receptors (GPCRs) is becoming increasingly important, both as a fundamental problem in membrane biology and as a consequence of their central role as pharmacological targets. Their integral membrane nature and rather low natural abundance present many challenging problems. Using a recently developed technique, plasmon-waveguide resonance (PWR) spectroscopy, we investigated the structural changes accompanying the binding of ligands to the human delta-opioid receptor (hDOR) immobilized in a solid-supported lipid bilayer. This highly sensitive technique can directly monitor changes in mass density, conformation, and orientation occurring in such thin proteolipid films. Without requiring labeling protocols, PWR allows the direct determination of binding constants in a system very close to the receptor's natural environment. In the present study, conformational changes of a proteolipid membrane containing the hDOR were investigated upon binding of a variety of peptide and nonpeptide agonists, partial agonists, antagonists, and inverse agonists. Distinctly different structural states of the membrane were observed upon binding of each of these classes of ligands, reflecting different receptor conformational states, and the formation of each state was characterized by different kinetic properties. Binding constants, obtained by quantifying the extent of conformational change as a function of the amount of ligand bound, were in good agreement with published values determined by radiolabeling methods. The results provide new insights into ligand-induced GPCR functioning and illustrate a powerful new protocol for drug development.
Asunto(s)
Antagonistas de Narcóticos/farmacología , Proteolípidos/efectos de los fármacos , Receptores Opioides delta/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetinae , Humanos , Ligandos , Membrana Dobles de Lípidos , Proteolípidos/metabolismo , Receptores Opioides delta/efectos de los fármacos , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.