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1.  Plasmon-waveguide resonance studies of ligand binding to integral proteins in membrane fragments derived from bacterial and mammalian cells 
Analytical biochemistry  2009;387(1):95-101.
A procedure has been developed for directly depositing membrane fragments derived from bacterial (chromatophores from Rhodopseudomonas sphaeroides) and mammalian cells (μ-opioid receptor- and MC4 receptor-transfected HEK cells, and rat trigeminal ganglion cells) onto the silica surface of a plasmon-waveguide resonance (PWR) spectrometer. Binding of ligands (cytochrome c2 for the chromatophores, the peptide agonists DAMGO and Melanotan-II that are specific for the μ-opioid and MC4 receptors, and two non-peptide agonists that are specific for the CB1 receptor) to these membrane fragments has been observed and characterized with high sensitivity using PWR spectral shifts. The KD values obtained are in excellent agreement with conventional pharmacological assays and with prior PWR studies using purified receptors inserted into deposited lipid bilayer membranes. These studies provide a new tool for obtaining useful biological information about receptor-mediated processes in real biological membranes.
doi:10.1016/j.ab.2009.01.019
PMCID: PMC2783692  PMID: 19454250
G-protein coupled receptors; bacterial chromatophores; transfected HEK cells; rat trigeminal ganglion; μ-opioid receptor; cannabinoid CB1 receptor; melanocortin-4 receptor
2.  Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling 
European journal of pharmacology  2007;581(1-2):19-29.
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 hCB1 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.
doi:10.1016/j.ejphar.2007.11.053
PMCID: PMC2279194  PMID: 18162180
trafficking; G proteins; PWR spectroscopy; functional selectivity
3.  The Two NK-1 Binding Sites Correspond to Distinct, Independent, and Non-Interconvertible Receptor Conformational States As Confirmed by Plasmon-Waveguide Resonance Spectroscopy 
Biochemistry  2006;45(16):5309-5318.
Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand–receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O2)11]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (Kd1 = 0.14 ± 0.02 nM and Kd2 = 1.4 ± 0.18 nM, and Kd1 = 5.5 ± 0.7 nM and Kd2 = 620 ± 117 nM, respectively), whereas only one binding affinity (Kd = 5.5 ± 0.4 nM) could be observed for propionyl[Met(O2)11]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).
doi:10.1021/bi052586d
PMCID: PMC1865500  PMID: 16618119
4.  New Paradigms and Tools in Drug Design for Pain and Addiction 
The AAPS journal  2006;8(3):E450-E460.
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and δ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
doi:10.1208/aapsj080353
PMCID: PMC1764851  PMID: 17025262
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors
5.  New paradigms and tools in drug design for pain and addiction 
The AAPS Journal  2006;8(3):E450-E460.
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and σ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in g-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
doi:10.1208/aapsj080353
PMCID: PMC1764851  PMID: 17025262
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors
6.  LIGAND MODULATION OF GPCR LATERAL SEGREGATION INTO LIPID MICRODOMAINS IN SPHINGOMYELIN/PHOSPHATIDYLCHOLINE SOLID-SUPPORTED BILAYERS 
Biochemistry  2005;44(25):9168-9178.
Increasing evidence supports the idea that the plasma membrane bilayer is characterized by a laterally inhomogeneous mixture of lipids, having an organized structure in which lipid molecules segregate into small domains or patches. Such microdomains are characterized by high contents of sphingolipids that form thicker liquid-ordered regions having resistance to extraction with nonionic detergents. The existence of lipid lateral segregation has been demonstrated in both model and biological membranes, although its role in protein sorting and membrane function still remains unclear. In the present studies, plasmon-waveguide resonance (PWR) spectroscopy was employed to investigate the properties of microdomains in a model system consisting of a solid-supported lipid bilayer composed of a 1:1 mixture of palmitoyloleoylphosphatidylcholine (POPC) and brain sphingomyelin (SM), and their influence on the partitioning and functioning of the human delta opioid receptor (hDOR; a G-protein coupled receptor, GPCR). Resonance signals corresponding to two microdomains (POPC-rich and SM-rich) were observed in such bilayers, and the sorting of the receptor into each domain was highly dependent on the type of ligand that was bound. When no ligand was bound, the receptor incorporated preferentially into the POPC-rich domain; when an agonist or an antagonist was bound, the receptor incorporated preferentially into the SM-rich component, although with a two-fold greater propensity for this microdomain in the case of the agonist. G-protein binding to the agonist-bound receptor in the SM-rich domain occurred with a 30-fold higher affinity than to the receptor in the PC-rich domain. The binding of agonist to an unliganded receptor in the bilayer produced receptor trafficking from the PC-rich into the SM-rich component. Since the SM-rich domain is thicker than the PC-rich domain, and previous studies with the hDOR have shown that the receptor elongates upon agonist-activation, we propose that hydrophobic matching between the receptor and the lipid is a driving force for receptor trafficking to the SM-rich component.
doi:10.1021/bi050207a
PMCID: PMC1440485  PMID: 15966741
microenvironmental effects; G-protein binding and activation; phospholipids; sphingolipids; protein sorting

Results 1-6 (6)