Rhodopsin is a member of an ancient class of receptors that transduce signals through their interaction with guanine nucleotide-binding proteins (G proteins). We have mapped the sites of interaction of rhodopsin with its G protein, which by analogy suggests how other members of this class of receptors may interact with their G proteins. Three regions of rhodopsin's cytoplasmic surface interact with the rod cell G protein transducin (Ga. These are (i) the second cytoplasmic loop, which connects rhodopsin helices m and IV, (U) the third cytoplasmic loop, which connects rhodopsin helices V and VI, and (iu) a putative fourth cytoplasmic loop formed by amino acids 310-321, as the carboxyl-terminal sequence emerges from helix VII and anchors to the lipid bilayer via palmitoylcysteines 322 and 323. Evidence for these regions of interaction of rhodopsin and Gt comes from the ability of synthetic peptides comprising these regions to compete with metarhodopsin II for binding to Gt. A spectroscopic assay that measures the "extra MIU" caused by Gt binding was used to measure the extent of binding of Gt in the presence of competing peptides. The three peptides corresponding to the second, third, and fourth cytoplasmic loops competed effectively with metarhodopsin H, exhibiting Kd values in the 2 jtM range; 11 additional peptides comprising all remaining surface regions of rhodopsin failed to compete even at 200 ,M. Any two peptides that were effective competitors showed a synergistic effect, having 15 times higher effectiveness when mixed than when assayed separately. A mathematical model was developed to describe this behavior.Rhodopsin is the best-studied receptor protein of that class of signal-transducing receptors that act via guanine nucleotidebinding proteins, or G proteins. Other members of this class include the adrenergic receptors (1), the muscarinic acetylcholine receptors (2), the substance K receptor (3) Gt. We previously showed (9) that selected peptides from the sequence of the Gt a subunit (Gta) can interfere with binding of Gt to photolyzed rhodopsin, thus allowing assignment of these peptides to the region of the Gta sequence that binds to rhodopsin. In the work described here, we tested peptides from the rhodopsin sequence in order to see which ones reduce the level of extra MII. Such peptides presumably would do so by simulating a region ofrhodopsin's surface that interacts with Gt, thus interfering with Gt binding to MII. MATERIALS AND METHODSSpectrophotometric Assay. Binding of Gt to MII was measured as in refs. 7-9. The assay was performed at pH 8 and 4°C, conditions under which only a small, control amount of MII is formed in the absence of Gt. The full extra MII signal in the presence of Gt corresponds to a 60% MII fraction ofthe total photoexcited rhodopsin. The final levels of MII formation minus the control level (no Gt present) are a direct measure of the rhodopsin-Gt complexes formed. When normalized to the undisturbed full extra MII signal, they yield the relative amount of Gt that is ...
Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.
Hydration properties of mixtures of a zwitterionic lipid, palmitoyloleoylphosphatidylcholine (POPC), and nonionic surfactants (oligo(oxyethylene) dodecyl ether, C12En with n ) 1-8) were studied over a wide range of surfactant/lipid molar ratios RA/L from 0.1 to 2 at T ) 25°C. The adsorption of water by the POPC/C12En mixtures was measured by the isopiestic method at two different relative humidities (RH ) 86.5 and 97%). Deuterium NMR on 2 H2O and 31 P NMR on the phospholipid as well as X-ray diffraction were employed to characterize the phase state of the mixtures. For samples in the LR phase the area requirement of POPC and surfactant molecules and the thickness of the hydrophobic core of the bilayer were estimated from the repeat spacing and the known composition of the sample. The experimental results are compared to data reported previously for pure POPC and C 12En systems under identical conditions. Small C12En concentrations (RA/L ) 0.1 and 0.2) in the membrane tighten the membrane packing. The area per molecule in the bilayer/water interface occupied by the lipid is reduced and that of the surfactant enlarged in the mixture compared to bilayers of the pure components under equal conditions. Further increase of the surfactant concentration causes a significant thinning of the hydrophobic core and a progressive increase of the area requirement of the amphiphilic molecules in the membrane/water interface. Finally, at high surfactant concentrations (R A/L ) 1 and 2) the area requirement of the amphiphilic constituents and the vertical extension of the polar interface region increase with growing ethylene oxide chain length n. The hydration of the lipid is reduced by the presence of C12En to a level comparable to the primary hydration shell. The first two or three oxyethylene groups next to the alkyl chain of the surfactant also show reduced hydration in the mixtures. The remaining EO groups have hydration characteristics very similar to the pure surfactant, with the exception of bilayers with RA/L ) 2 at RH ) 97%.
We have developed a reusable piezoelectric immunosensor for the detection of the following human herpes viruses: herpes simplex viruses types 1 and 2, varicella-zoster virus, Epstein-Barr virus, and cytomegalovirus. Synthetic peptides, representing different surface antigens of the five viruses, were used to generate virus-specific monoclonal antibodies. Apply an antibody layer via protein A immobilization onto a 10-MHz AT-cut crystal resulted in 5 x 10(4)-1 x 10(9) viruses on the electrode surface in a linear frequency change and a long-term stability of 8 weeks when the modified crystal was stored dry over silica gel blue at room temperature. Under these conditions, the coated crystal can be used 18 times without detectable loss of activity.
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