Binding of agonists, antagonists and inverse agonists to the human delta-opioid receptor produces distinctly different conformational states distinguishable by plasmon-waveguide resonance spectroscopy.

Structural changes induced by the binding of agonists, antagonists and inverse agonists to the cloned delta-opioid receptor from human brain immobilized in a solid-supported lipid bilayer were monitored using plasmon-waveguide resonance (PWR) spectroscopy. Agonist (e.g. deltorphin II) binding causes an increase in membrane thickness because of receptor elongation, a mass density increase due to an influx of lipid molecules into the bilayer, and an increase in refractive index anisotropy due to transmembrane helix and fatty acyl chain ordering. In contrast, antagonist (e.g. TIPPpsi) binding produces no measurable change in either membrane thickness or mass density, and a significantly larger increase in refractive index anisotropy, the latter thought to be due to a greater extent of helix and acyl chain ordering within the membrane interior. These results are closely similar to those reported earlier for another agonist (DPDPE) and antagonist (naltrindol) [Salamon et al. (2000) Biophys. J.79, 2463-2474]. In addition, we now find that an inverse agonist (TMT-Tic) produces membrane thickness, mass density and refractive index anisotropy increases which are similar to, but considerably smaller than, those generated by agonists. Thus, a third conformational state is produced by this ligand, different from those formed by agonists and antagonists. These results shed new light on the mechanisms of ligand-induced G-protein-coupled receptor functioning. The potential utilization of this new biophysical method to examine structural changes both parallel and perpendicular to the membrane normal for GPCRs is emphasized.     

Phosphatidylethanolamine enhances rhodopsin photoactivation and transducin binding in a solid supported lipid bilayer as determined using plasmon-waveguide resonance spectroscopy

Flash photolysis studies have shown that the membrane lipid environment strongly influences the ability of rhodopsin to form the key metarhodopsin II intermediate. Here we have used plasmon-waveguide resonance (PWR) spectroscopy, an optical method sensitive to both mass and conformation, to probe the effects of lipid composition on conformational changes of rhodopsin induced by light and due to binding and activation of transducin (G(t)). Octylglucoside-solubilized rhodopsin was incorporated by detergent dilution into solid-supported bilayers composed either of egg phosphatidylcholine or various mixtures of a nonlamellar-forming lipid (dioleoylphosphatidylethanolamine; DOPE) together with a lamellar-forming lipid (dioleoylphosphatidylcholine; DOPC). Light-induced proteolipid conformational changes as a function of pH correlated well with previous flash photolysis studies, indicating that the PWR spectral shifts monitored metarhodopsin II formation. The magnitude of these effects, and hence the extent of the conformational transition, was found to be proportional to the DOPE content. Our data are consistent with previous suggestions that lipids having a negative spontaneous curvature favor elongation of rhodopsin during the activation process. In addition, measurements of the G(t)/rhodopsin interaction in a DOPC/DOPE (25:75) bilayer at pH 5 demonstrated that light activation increased the affinity for G(t) from 64 nM to 0.7 nM, whereas G(t) affinity for dark-adapted rhodopsin was unchanged. By contrast, in DOPC bilayers the affinity of G(t) for light-activated rhodopsin was only 18 nM at pH 5. Moreover exchange of GDP for GTP gamma S was also monitored by PWR spectroscopy. Only the light-activated receptor was able to induce this exchange which was unaffected by DOPE incorporation. These findings demonstrate that nonbilayer-forming lipids can alter functionally linked conformational changes of G-protein-coupled receptors in membranes, as well as their interactions with downstream effector proteins.     

Direct nuclear magnetic resonance observation of odorant binding to mouse odorant receptor MOR244-3

Mammals are able to perceive and differentiate a great number of structurally diverse odorants through the odorant's interaction with odorant receptors (ORs), proteins found within the cell membrane of olfactory sensory neurons. The natural gas industry has used human olfactory sensitivity to sulfur compounds (thiols, sulfides, etc.) to increase the safety of fuel gas transport, storage, and use through the odorization of this product. In the United States, mixtures of sulfur compounds are used, but the major constituent of odorant packages is 2-methylpropane-2-thiol, also known as tert-butyl mercaptan. It has been fundamentally challenging to understand olfaction and odorization due to the low affinity of odorous ligands to the ORs and the difficulty in expressing a sufficient number of OR proteins. Here, we directly observed the binding of tert-butyl mercaptan and another odiferous compound, cis-cyclooctene, to mouse OR MOR244-3 on living cells by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy. This effort lays the groundwork for resolving molecular mechanisms responsible for ligand binding and resulting signaling, which in turn will lead to a clearer understanding of odorant recognition and competition.     

Ligand binding profiles of delta-opioid receptor in human cerebral cortex membranes: evidence of delta-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.     

The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy

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(O(2))(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 (K(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 5.5 +/- 0.7 nM and K(d2) = 620 +/- 117 nM, respectively), whereas only one binding affinity (K(d) = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O(2))(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).     

Plasmon resonance studies of agonist/antagonist binding to the human delta-opioid receptor: new structural insights into receptor-ligand interactions

Structural changes accompanying the binding of ligands to the cloned human delta-opioid receptor immobilized in a solid-supported lipid bilayer have been investigated using coupled plasmon-waveguide resonance spectroscopy. This highly sensitive technique directly monitors mass density, conformation, and molecular orientation changes occurring in anisotropic thin films and allows direct determination of binding constants. Although both agonist binding and antagonist binding to the receptor cause increases in molecular ordering within the proteolipid membrane, only agonist binding induces an increase in thickness and molecular packing density of the membrane. This is a consequence of mass movements perpendicular to the plane of the bilayer occurring within the lipid and receptor components. These results are consistent with models of receptor function that involve changes in the orientation of transmembrane helices.     

Direct coupling of a G-protein to dihydropyridine binding sites

Electrophysiological data support the existence of GTP-binding proteins interacting with voltage dependent calcium channels. Along this line the present study investigates the effect of GMP-PNP, a stable GTP analogue, on the displacement of [3H]-PN 200-110 binding by agonist and antagonist dihydropyridines in synaptic membranes prepared from rat cortex. The results show that GMP-PNP increases the ability of the agonist dihydropyridine BAY K 8644 to displace [3H]-PN 200-110 binding. The in vivo treatment with Pertussis Toxin abolishes the effect produced by the non-hydrolysable GTP analogue.     

Exploring deltorphin II binding to the third extracellular loop of the delta-opioid receptor

The third extracellular loop of the human delta-opioid receptor (hDOR) is known to play an important role in the binding of delta-selective ligands. In particular, mutation of three amino acids (Trp(284), Val(296), and Val(297)) to alanine significantly diminished delta-opioid receptor affinity for delta-selective ligands. To assess the changes in conformation accompanying binding of the endogenous opioid peptide deltorphin II to the delta-opioid receptor at both the receptor and ligand levels as well as to determine points of contact between the two, an in-depth spectroscopic study that addressed these points was initiated. Fragments of the delta-opioid receptor of variable length and containing residues in the third extracellular loop were synthesized and studied by NMR and CD spectroscopy in a membrane-mimetic milieu. The receptor peptides examined included hDOR-(279-299), hDOR-(283-299), hDOR-(281-297), and hDOR-(283-297). A helical conformation was observed for the longest receptor fragment between Val(283) and Arg(291), whereas a nascent helix occurred in a similar region for hDOR-(281-297). Further removal of N-terminal residues Val(281) and Ile(282) abolished helical conformation completely. Binding of the delta-selective ligand deltorphin II to hDOR-(279-299) destabilized the helix at the receptor peptide N terminus. Dramatic changes in the alpha-proton chemical shifts for Trp(284) and Leu(286) in hDOR-(279-299) also accompanied this loss of helical conformation. Large upfield displacement of alpha-proton chemical shifts was observed for Leu(295), Val(296), and Val(297) in hDOR-(279-299) following its interaction with deltorphin II, thus identifying a gain in beta-conformation at the receptor peptide C terminus. Similar changes did not occur for the shorter peptide hDOR(281-297). A hypothesis describing the conformational events accompanying selective deltorphin II binding to the delta-opioid receptor is presented.     

Binding of oxidized and reduced cytochrome c2 to photosynthetic reaction centers: plasmon-waveguide resonance spectroscopy

The dissociation constants for the binding of oxidized and reduced wild-type cytochrome c(2) from Rhodobacter capsulatus and the lysine 93 to proline mutant of cytochrome c(2) to photosynthetic reaction centers (Rhodobacter sphaeroides) has been measured to high precision using plasmon-waveguide resonance spectroscopy. For the studies reported, detergent-solubilized photosynthetic reaction center was exchanged into a phosphatidylcholine lipid bilayer to approximate the physiological environment. At physiologically relevant ionic strengths ( approximately 100 mM), we found two binding sites for the reduced wild-type cytochrome (K(D) = 10 and 150 nM), with affinities that decrease with decreasing ionic strength (2-5-fold). These results implicate nonpolar interactions as an important factor in determining the dissociation constants. Taking advantage of the ability of plasmon-waveguide resonance spectroscopy to reslove the contribution of changes in mass and of structural anisotropy to cytochrome binding, we can demonstrate very different properties for the two binding sites. In contrast, the oxidized wild-type cytochrome only binds to a single site with a K(D) of 10 nM at high ionic strength, and this site has properties similar to the low-affinity site for binding the reduced cytochrome. The binding of oxidized cytochrome c(2) has a strong ionic strength response, with the affinity decreasing approximately 30-fold in going from high to low ionic strength. The K93P mutant binds to a single site in both redox states, which is similar, in terms of mass and structural anisotropy, to the oxidized wild-type site, with the affinity of the mutant oxidized state being approximately 30-fold weaker than that of the oxidized wild-type cytochrome at high ionic strength. Thus, reduced wild-type cytochrome can bind to both the high- and low-affinity sites, while the oxidized wild-type cytochrome and both redox states of the mutant cytochrome can only bind to the low-affinity site, possibly the consequence of the more stable structure of reduced wild-type cytochrome. In aggregate, the results are consistent with a model in which a transient conformational change in the region 88-102 in the cytochrome three-dimensional structure, the so-called hinge region, drives the dissociation of the oxidized cytochrome from the reaction center-cytochrome complex, facilitating turnover.     

Direct observation of active protein folding using lock-in force spectroscopy

Direct observation of the folding of a single polypeptide chain can provide important information about the thermodynamic states populated along its folding pathway. In this study, we present a lock-in force-spectroscopy technique that improves resolution of atomic-force microscopy force spectroscopy to 400 fN. Using this technique we show that immunoglobulin domain 4 from Dictyostelium discoideum filamin (ddFLN4) refolds against forces of ∼4 pN. Our data show folding of this domain proceeds directly from an extended state and no thermodynamically distinct collapsed state of the polypeptide before folding is populated. Folding of ddFLN4 under load proceeds via an intermediate state. Three-state folding allows ddFLN4 to fold against significantly larger forces than would be possible for a mere two-state folder. We present a general model for protein folding kinetics under load that can predict refolding forces based on chain-length and zero force refolding rate.     

Picosecond dynamics of G-protein coupled receptor activation in rhodopsin from time-resolved UV resonance Raman spectroscopy

The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the beta-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsin-to-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.     

Direct observation of fibrinogen-heparinoid complexes formation using surface plasmon resonance.

We analyzed the binding of heparinoid or heparin with fibrinogen by real-time measurement using surface plasmon resonance technology. Poly(glucosyloxyethyl methacrylate) sulfate [poly(GEMA) sulfate] and dextran sulfate were used as heparinoids. The binding ability of each sulfated polymer was estimated by having each polymer-containing buffer interact with the sensor chip surfaces that had immobilized fibrinogen. Dextran sulfate and poly(GEMA) sulfate showed high affinity to the fibrinogen in this experiment, while the heparin did not. All of the dextran sulfates were desorbed from its surface, while about 30% of the poly(GEMA) sulfate remained on the immobilized fibrinogen upon the addition of NaCl to the buffer which was done in order to analyze the desorption of poly(GEMA) sulfate or dextran sulfate from the surface of the fibrinogen. These data show that the type of binding between fibrinogen-poly(GEMA) sulfate was different from that of dextran sulfate, indicating that the interaction between fibrinogen and poly(GEMA) sulfate was caused not only by an electrostatic but also by a hydrophobic force. These results suggest that the interaction mechanism of heparinoids with fibrinogen was different from that of heparin.     

Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor

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.     

Direct observation of the methionine residues of cytochrome c by 13C nuclear magnetic resonance spectroscopy.

The two Cepsilon-methyl methionine groups in cytochrome c have been chemically enriched (45%) with 13C. Their 13C NMR signals have been monitored in both the oxidized and reduced states and under various solution conditions. Methionine residue 80 showed characteristic chemical shift positions for the reduced Fe(II) and cyano-Fe(III) forms. No signal for methionine 80 was observed in the oxidized Fe(III) form due to the paramagnetic effect of the iron atom to which it is bonded, but the position of the methionine 65 signal was shifted, indicating that it is sensitive to the change of oxidation state. Two well resolved signals were observed at pH 11 for the Fe(III) form but only one was resolved at pH 2, indicating that while methionine 80 is definitely displaced from the iron atom at alkaline pH, it may not be in acid conditions.     

Direct observation to chemokine receptor 5 on T-lymphocyte cell surface using fluorescent metal nanoprobes

Chemokine receptor 5 (CCR5) is a cell surface protein required for HIV-1 infection. It is important to detect the amount and observe the spatial distribution of the CCR5 receptors on the cell surfaces. In this report, we describes the metal nanoparticles which were specially designed as molecular fluorescent probes for imaging of CCR5 receptors on the T-lymphocytic PM1 cell surfaces. These CCR5 monoclonal antibodies (mAbs) metal complexes were prepared by labeling mAbs with Alexa Fluor 680 followed by covalent binding the labeled mAbs on the 20 nm silver nanoparticles. Compared with the labeled mAbs without metal, the mAb-metal complexes were found to display enhanced emission intensity and shortened lifetime due to interactions between fluorophores and metal. The mAb-metal complexes were incubated with the PM1 cell lines. The confocal fluorescent intensity and lifetime cell images were recorded on single cells. It was observed that the mAb-metal complexes could be clearly distinguished from the cellular autofluorescence. By analyzing a pool of cell images, we observed that most CCR5 receptors appeared as clusters on the cell surfaces. The fluorophore-metal complexes developed in this report are generally useful for detection of cell surface receptors and provide a new class of probe to study the interaction between the CCR5 receptors with viral gp120 during HIV infections.     

Interaction of phosphatidylserine synthase from E. coli with lipid bilayers: coupled plasmon-waveguide resonance spectroscopy studies

The interaction of phosphatidylserine (PS) synthase from Escherichia coli with lipid membranes was studied with a recently developed variant of the surface plasmon resonance technique, referred to as coupled plasmon-waveguide resonance spectroscopy. The features of the new technique are increased sensitivity and spectral resolution, and a unique ability to directly measure the structural anisotropy of lipid and proteolipid films. Solid-supported lipid bilayers with the following compositions were used: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA) (80:20, mol/mol); POPC-POPA (60:40, mol/mol); and POPC-1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) (75:25, mol/mol). Addition of either POPA or POPG to a POPC bilayer causes a considerable increase of both the bilayer thickness and its optical anisotropy. PS synthase exhibits a biphasic interaction with the bilayers. The first phase, occurring at low protein concentrations, involves both electrostatic and hydrophobic interactions, although it is dominated by the latter, and the enzyme causes a local decrease of the ordering of the lipid molecules. The second phase, occurring at high protein concentrations, is predominantly controlled by electrostatic interactions, and results in a cooperative binding of the enzyme to the membrane surface. Addition of the anionic lipids to a POPC bilayer causes a 5- to 15-fold decrease in the protein concentration at which the first binding phase occurs. The results reported herein lend experimental support to a previously suggested mechanism for the regulation of the polar head group composition in E. coli membranes.     

The aspartic acid in deltorphin I and dermenkephalin promotes targeting to delta-opioid receptor independently of receptor binding.

Recent studies on the highly potent and selective delta-opioid agonists demenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2) and deltorphin I (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) suggested that key structural features necessary for specific targetting to the delta-opioid receptor are located within the C-terminal halves of these naturally occurring heptapeptides. To investigate the contribution of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin to the delta-addressing ability of the C-terminal ends, fourteen analogs were synthesized and assessed for their ability to bind to mu and delta-opioid receptors in rat brain membrane homogenates. Results showed that i/ although the tetrapeptide C-terminus of dermenkephalin and deltorphin I differ in amino acid composition, they play a similar role in specifying correct addressing of these peptides to the delta-receptor, ii/ the negatively charged side chain of aspartic acid 4 residue in deltorphin I and aspartic acid 7 residue in dermenkephalin is not involved in binding contact at the delta-receptor site, nor in maintaining a delta-bioactive folding of the peptides, iii/ these side chains are, in contrast, functionally or structurally required to confer high delta-selectivity by preventing mu-site recognition and/or binding.     

Direct observation of the titration of substrate carbonyl groups in the active site of alpha-chymotrypsin by resonance Raman spectroscopy

By use of resonance Raman (RR) spectroscopy, the population of the reactive carbonyl group in active acylchymotrypsins has been characterized and correlated with acyl-enzyme reactivity. RR spectra have been obtained, with a flow system and 324- and 337.5-nm excitation, at low and active pH for six acylchymotrypsins, viz., (indoleacryloyl)-, (4-amino-3-nitrocinnamoyl)-, (furylacryloyl)-, [( 5-ethylfuryl)-acryloyl]-, (thienylacryloyl)-, and [( 5-methylthienyl)acryloyl]chymotrypsin. These acyl-enzymes represent a 100-fold range of deacylation rate constants. Good RR spectral quality has enabled us to obtain the vibrational spectrum of the carbonyl group at low and active pH in each acyl-enzyme. The measured pKa of the spectroscopic changes in the carbonyl region is identical with that for the deacylation kinetics, showing that the RR carbonyl features reflect the ionization state of His-57. A carbonyl population has been observed in the active acyl-enzymes in which the carbonyl oxygen atom of the reactive acyl linkage is hydrogen-bonded in the active site. The proportion of this hydrogen-bonded population, with respect to other observed non-hydrogen-bonded species, together with the degree of polarization of the carbonyl bond, as monitored by vC = 0, has been correlated with the deacylation rate constants of the acyl-enzymes. It is proposed that the hydrogen-bonded carbonyl species is located at or near the oxyanion hole and represents the ground state from which deacylation occurs. An increase in the proportion of the hydrogen-bonded population and an increase in polarization of the carbonyl bond result in an increase in deacylation rate constant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-specific regulation of the delta-opioid receptor

Delta opioid receptor (DOR) agonists are attractive potential analgesics, since these compounds exhibit strong antinociceptive activity with relatively few side effects. In the past decade, several novel classes of delta-opioid agonists have been synthesized. Recent experimental data indicate that structurally distinct opioid agonists interact differently with the delta-opioid receptor. Consequently, individual agonist-bound DOR conformations may interact differently with intracellular proteins. In the present paper, after a brief review of the cellular processes that contribute to homologous desensitization of the DOR signaling, we shall focus on experimental data demonstrating that chemically different agonists differ in their ability to phosphorylate, internalize, and/or down-regulate the DOR. Homologous regulation of the opioid receptor signaling is thought to play an important role in the development of opioid tolerance. Therefore, agonist-specific differences in DOR regulation suggest that by further chemical modification, delta-selective opioid analgesics can be designed that exhibit a reduced propensity for analgesic tolerance.     

Direct sugar binding to LacY measured by resonance energy transfer

Trp151 in the lactose permease of Escherichia coli (LacY) is an important component of the sugar-binding site and the only Trp residue out of six that is in close proximity to the galactopyranoside in the structure (1PV7). The short distance between Trp151 and the sugar is favorable for F?rster resonance energy transfer (FRET) to nitrophenyl or dansyl derivatives with the fluorophore at the anomeric position of galactose. Modeling of 4-nitrophenyl-alpha-d-galactopyranoside (alpha-NPG) in the binding-site of LacY places the nitrophenyl moiety about 12 A away from Trp151, a distance commensurate with the F?rster distance for a Trp-nitrobenzoyl pair. We demonstrate here that alpha-NPG binding to LacY containing all six native Trp residues causes galactopyranoside-specific FRET from Trp151. Moreover, binding of alpha-NPG is sufficiently slow to resolve time-dependent fluorescence changes by stopped-flow. The rate of change in Trp --> alpha-NPG FRET is linearly dependent upon sugar concentration, which allows estimation of kinetic parameters for binding. Furthermore, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) covalently attached to the cytoplasmic end of helix X is sensitive to sugar binding, reflecting a ligand-induced conformational change. Stopped-flow kinetics of Trp --> alpha-NPG FRET and sugar-induced changes in MIANS fluorescence in the same protein reveal a two-step process: a relatively rapid binding step detected by Trp151 --> alpha-NPG FRET followed by a slower conformational change detected by a change in MIANS fluorescence.