A synthetic heptaphosphopeptide comprising the fully pbosphorylated carboxyl terminal pbosphorylation region of bovine rbodopsin, residues 330-348, was found to induce a conformational change in bovine arrestin. This caused an alteration of the pattern of limited proteolysis of arrestin similar to that induced by binding pbospborylated rbodopsin or heparin. Unlike heparin, the phosphopeptide also induced light-activated binding of arrestin to both unphospborylated rhodopsin in disk membranes as well as to endoproteinase Asp-N-treated rbodopsin (des 330-348). These findings suggest that one function of phosphorylation of rhodopsin is to activate arrestin which can then bind to other regions of the surface of the photoactivated rhodopsin. It has been recognized that phosphorylation of the activated receptor greatly enhances arrestin binding, and that binding is minimal to the unphosphorylated receptor [4,[6][7][8]. In the present work, we show that in the presence of a synthetic phosphorylated peptide from rhodopsin's carboxyl-terminal sequence, arrestin can bind to photoactivated unphosphorylated rhodopsin. We demonstrate that binding of the phosphopeptide to arrestin causes a change in the conformation of arrestin, and that it is this new conformation of arrestin that is capable of binding to photoactivated rhodopsin. Other G-proteinlinked receptors may similarly activate their corresponding arrestins which can then bind to other regions of the surface of the activated receptor.

Series of uniformly and selectively 15N‐labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H‐15N‐NMR study was performed in methanol/chloroform (1:1) and 0.1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross‐peaks expected from the amino acid sequence of uniformly 15N‐labeled bacteriorhodopsin were observed, using heteronuclear 1H‐15N coherence spectroscopy. In order to assign the observed cross‐peaks, a selective 15N‐labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H‐15N‐NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72–231), C2 (residues 1–71), B1 (residues 1–155) and BP2 (residues 163–231) were investigated. By this procedure, all observed 1H‐15N cross‐peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B and G. The cross‐peaks from four (C, D, E and F) helical bundles (79–189 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.

Proteolytic fragment 163 -231 of bacterioopsin was isolated from Halobacterium halobium purple membrane treated with NaBH, and papain under nondenaturing conditions. Two-dimensional 'H-NMR spectra of (163 -231)-bacterioopsin solubilized in chloroform/methanol (1 : I), 0.1 M LiC104 indicated the existence of one predominant conformation. Most of the resonances in the 'H-NMR spectra of (1 63 -231)-bacterioopsin were assigned by two-dimensional techniques. Two extended right-handed a-helical regions Ala168 -Ilel91 and Am202 -Arg227 were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the peptide is flexible and the region of Gly192-Leu201 adopts a specific conformation. The protons of OH groups of Thr178, Ser183 and Ser214 slowly exchange with solvent, and side-chain conformations of these residues, as evaluated by NOE connectivities of OH protons, are optimal for the formation of hydrogen bonds between OH and backbone carbonyl groups.Bacteriorhodopsin, a light-driven proton pump, is the main component of the purple membrane of Halobacterium halobium (reviewed in [l -31). It consists of 248 amino acid residues, most of which are embedded into the membrane. The light absorption by chromophore retinal bound to Lys216 [4] triggers the photocycle that leads to proton translocation across the membrane. Site-directed mutagenesis revealed some aromatic and charged amino acid residues thus affecting the proton translocation efficiency and/or the chromophore absorption maximum [5 -71. The detailed three-dimensional structure of bacteriorhodopsin will undoubtedly promote a better understanding of the mechanism of vectorial proton translocation.Bacteriorhodopsin readily forms stable and highly ordered two-dimensional crystals. Electron microscopy of such crystals revealed seven electron-dense membrane-spanning segments, probably being the seven a-helices A-G [8]. Proteolytic digestion, immunochemical methods and theoretical considerations [l -31 resulted in a rough location of transmembrane segments in the bacteriorhodopsin amino acid sequence.Two-dimensional (2D) 'H-NMR is an elaborate method for conformational analysis of polypeptides in solution [9]. Unfortunately, the proton resonances of bacteriorhodopsin in bilayer membranes or small liposomes are too broad to be resolved. Thus an artificial medium conserving the protein structure and providing high-resolution NMR spectra is the main hindrance when using this method to study membrane proteins. According to CD and "F-NMR spectroscopy, a chloroform/methanol mixture is a suitable medium [lo]. Bacteriorhodopsin and its proteolytic fragments, being This paper deals with the conformation of (163 -231)-bacterioopsin obtained by NaBH, and papain treatment of the purple membrane and then solubilized in the chloroform/ methanol mixture. Most of the resonance in the 'H-NMR spectra of (163 -231)-bacterioopsin were assigned. NOE connectivities and deuterium exchange rates underlay identification of two right-handed a-helical regions...

The sulfhydryl groups of the three cysteines in bovine arrestin react with DTNB very slowly (over a period of several hours). In the presence of the synthetic phosphopeptide comprising the fully phosphorylated carboxyl-terminal 19 amino acids of bovine rhodopsin, the reactivity of one of the sulfhydryls was enhanced while that of another was greatly reduced. Since this synthetic peptide was shown to activate arrestin with respect to its binding to unphosphorylated, light-activated rhodopsin, the reactivity of the sulfhydryl groups of a constitutively active R175Q arrestin mutant was examined. All three of the sulfhydryl groups of the mutant arrestin R175Q reacted rapidly with DTNB, but not as rapidly as with SDS-denatured arrestin. The arrestin mutant R175Q bound to light-activated, unphosphorylated rhodopsin in ROS disk membranes. The arrestin mutant R175Q also inhibited the light-activated PDE activity with an IC50 of 1.3 microM under the experimental conditions that were used. These data indicate that each of these forms of arrestin is a different conformation. The activated conformation of arrestin that binds to phosphorylated rhodopsin in vivo may be yet another conformation. We conclude that arrestin is a flexible molecule that is able to attain several different conformations, all of which are able to attain the activated functional state of arrestin.

The conformation of chymotryptic fragment C2 of bacteriohodopsin (residues 1-71) was studied by 2D 1H NMR. The fragment was solubilized in a mixture of chloroform/methanol (1:1), 0.1 M LiClO4. Most of the resonances in 1H NMR spectra of fragment C2 were assigned using phase-sensitive DQF-COSY, TOCSY, and NOESY techniques. To simplify the assignment procedure for overlapping regions of NMR spectra, an analog of fragment C2 with leucines deuterated in beta-positions was used. Deuterium exchange rates for amide protons were measured in a series of TOCSY spectra. Two right-handed alpha-helical regions Pro8-Lys30 and Lys41-Leu62 were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the fragment (Ala2-Gly6) adopts the helical conformation stabilized by 3 hydrogen bonds.