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• W2037594168 abstract "We report that acidic phospholipids can restore the binding of visual arrestin to purified rhodopsin solubilized in n-dodecyl-β-d-maltopyranoside. We used this finding to investigate the interplay between arrestin binding and the status of the retinal chromophore ligand in the receptor binding pocket. Our results showed that arrestin can interact with the late photoproduct Meta III and convert it to a Meta II-like species. Interestingly in these mixed micelles, the release of retinal and arrestin was no longer directly coupled as it is in the native rod disk membrane. For example, up to ∼50% of the retinal could be released even though arrestin remains bound to the receptor in a long lived complex. We anticipate that this new ability to study these proteins in a defined, purified system will facilitate further structural and dynamic studies of arrestin-rhodopsin interactions. We report that acidic phospholipids can restore the binding of visual arrestin to purified rhodopsin solubilized in n-dodecyl-β-d-maltopyranoside. We used this finding to investigate the interplay between arrestin binding and the status of the retinal chromophore ligand in the receptor binding pocket. Our results showed that arrestin can interact with the late photoproduct Meta III and convert it to a Meta II-like species. Interestingly in these mixed micelles, the release of retinal and arrestin was no longer directly coupled as it is in the native rod disk membrane. For example, up to ∼50% of the retinal could be released even though arrestin remains bound to the receptor in a long lived complex. We anticipate that this new ability to study these proteins in a defined, purified system will facilitate further structural and dynamic studies of arrestin-rhodopsin interactions. The integral membrane protein rhodopsin (Rho) 2The abbreviations used are: Rho, rhodopsin; Rho-P, phosphorylated rhodopsin; Rho*, light-activated rhodopsin; Meta I, metarhodopsin I; Meta II, metarhodopsin II; Meta III, metarhodopsin III; ROS, rod outer segment or wild-type rhodopsin in native membranes; ROS-P, phosphorylated ROS; DM; n-dodecyl-β-d-maltopyranoside; PC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; PS, 1,2-dioleoyl-sn-glycero-3-phosphoserine; PI, l-α-phosphatidylinositol; PA, 1,2-dioleoyl-sn-glycero-3-phosphate; λmax, wavelength of maximum absorption; λex, wavelength of excitation; λem, wavelength of emission; MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; I72B, bimane-labeled arrestin I72C/W194F. 2The abbreviations used are: Rho, rhodopsin; Rho-P, phosphorylated rhodopsin; Rho*, light-activated rhodopsin; Meta I, metarhodopsin I; Meta II, metarhodopsin II; Meta III, metarhodopsin III; ROS, rod outer segment or wild-type rhodopsin in native membranes; ROS-P, phosphorylated ROS; DM; n-dodecyl-β-d-maltopyranoside; PC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; PS, 1,2-dioleoyl-sn-glycero-3-phosphoserine; PI, l-α-phosphatidylinositol; PA, 1,2-dioleoyl-sn-glycero-3-phosphate; λmax, wavelength of maximum absorption; λex, wavelength of excitation; λem, wavelength of emission; MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; I72B, bimane-labeled arrestin I72C/W194F. enables the conversion of light to nerve signals in the rod cells, resulting in dim light vision (1Pepe I.M. Prog. Retin. Eye Res. 2001; 20: 733-759Crossref PubMed Scopus (58) Google Scholar, 2Lamb T.D. Pugh Jr., E.N. Prog. Retin. Eye Res. 2004; 23: 307-380Crossref PubMed Scopus (509) Google Scholar, 3Ridge K.D. Abdulaev N.G. Sousa M. Palczewski K. Trends Biochem. Sci. 2003; 28: 479-487Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). In the dark, the chromophore (11-cis-retinal) is linked to Rho at Lys296 by a protonated Schiff base (λmax ∼ 500 nm). Light absorption isomerizes the chromophore to all-trans-retinal. Within milliseconds two photoproducts evolve, Meta I (λmax ∼ 480 nm) and Meta II (λmax ∼ 380 nm), which are in a pH- and temperature-sensitive equilibrium. The ability of Meta II to bind and activate the G-protein transducin (4Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar) is terminated in several ways. Meta II can decay through hydrolysis of the Schiff base linkage and release of retinal, a process that takes ∼1 min at physiological temperature and pH (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Alternatively Meta II can decay to the long lived retinal storage photoproduct Meta III (λmax ∼ 470 nm) in which the Schiff base is intact and protonated (6Heck M. Schadel S.A. Maretzki D. Bartl F.J. Ritter E. Palczewski K. Hofmann K.P. J. Biol. Chem. 2003; 278: 3162-3169Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Vogel R. Siebert F. Mathias G. Tavan P. Fan G. Sheves M. Biochemistry. 2003; 42: 9863-9874Crossref PubMed Scopus (44) Google Scholar, 8Vogel R. Siebert F. Zhang X.Y. Fan G. Sheves M. Biochemistry. 2004; 43: 9457-9466Crossref PubMed Scopus (29) Google Scholar, 9Lewis J.W. van Kuijk F.J. Carruthers J.A. Kliger D.S. Vision Res. 1997; 37: 1-8Crossref PubMed Scopus (37) Google Scholar). Finally Rho signaling can be blocked through a series of protein-protein interactions that involves phosphorylation of the C-terminal tail of Rho by Rho kinase and binding of the protein arrestin (10Gurevich V.V. Gurevich E.V. Trends Pharmacol. Sci. 2004; 25: 105-111Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). Earlier we found that these inactivation mechanisms are related. That is, arrestin release and retinal release appear to be directly linked events: both are described by similar activation energies, and arrestin slows the rate of retinal release ∼2-fold at physiological temperatures. Intriguingly we also found that a fraction of the arrestin remains bound to ROS*-P long after “active” Meta II decay (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In the present work, we expanded upon these studies and made several surprising discoveries. First, we found that adding phospholipids restores arrestin binding to purified Rho*-P solubilized in n-dodecyl-β-d-maltopyranoside (DM). Second, we clearly established in the mixed micelle system that arrestin interacts with the post-Meta II photodecay product Meta III. Finally we found that arrestin and retinal release appear to be unlinked in mixed micelles with the acidic phospholipids PS, PI, and PA showing a more pronounced effect than the neutral phospholipids PC and PE (Fig. 1 shows a scheme of the different lipid head groups). Intriguingly with the acidic phospholipids, arrestin dissociation was nearly completely inhibited, yet approximately half of the retinal was released with the remainder trapped in the binding pocket. Materials—Frozen bovine retinas were purchased from Lawson and Lawson, Inc. (Lincoln, NE), and GBX red light filters were from Eastman Kodak Co. 11-cis-Retinal was a generous gift from Rosalie Crouch (Medical University of South Carolina and NEI, National Institutes of Health). Biomax centrifugal concentrators (10-kDa cutoff) were obtained from Millipore (Bedford, MA), and monobromobimane was purchased from Molecular Probes (Eugene, OR). Cuvettes were purchased from Uvonics (Plainview, NY), and band pass filters and long pass filters were obtained from Oriel (Stratford, CT). Acrylamide/bisacrylamide solution (37.5:1), and microcolumns were purchased from Bio-Rad. Concanavalin A-Sepharose, HiTrap heparin, and HiTrap Q prepacked columns were obtained from Amersham Biosciences. Spectroscopic grade buffers were from U. S. Biochemical Corp. (Cleveland, OH). Asolectin was purchased from Fluka (Buchs, Switzerland), and DM was from Anatrace (Maumee, OH). Purified phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), and 1,2-dioleoyl-sn-glycero-3-phosphoserine (PS), l-α-phosphatidylinositol (PI, from soy), and 1,2-dioleoyl-sn-glycero-3-phosphate (PA) were obtained from Avanti Polar Lipids (Alabaster, AL). All other chemicals and reagents were purchased from Sigma. Buffers—Buffer A consisted of 137 mm NaCl, 2.7 mm KCl, 1.5 mm KH2PO4, 8 mm NaHPO4, 2 mm CaCl2, 2 mm MgCl2, 2 mm MnCl2, pH 6.0. Buffer B consisted of 20 mm HEPES, 2 mm CaCl2, 2 mm MgCl2, 2 mm MnCl2, pH 6.5. Buffer C consisted of 20 mm HEPES, pH 7.4. Buffer D consisted of 10 mm Tris-HCl, 2 mm EDTA, 100 mm NaCl, pH 7.5. Buffer E consisted of 10 mm Tris-HCl, 2 mm EDTA, pH 7.0. Buffer F consisted of 10 mm Tris-HCl, 2 mm EDTA, pH 8.5. Buffers A, B, and C were supplemented with 0.1 mm phenylmethylsulfonyl fluoride, and Buffers D, E, and F were supplemented with 1 mm dithiothreitol and protease inhibitor mixture (Sigma, for bacterial cell extracts) immediately before use. Preparation of Rod Outer Segments and Purification of Rho—ROS and highly phosphorylated ROS were prepared from bovine retinas as described previously (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Rho was purified using concanavalin A. Briefly ROS containing 1 mg of Rho was solubilized in 14 ml of Buffer A containing 1% DM (mixing for 30 min at 4 °C) and then clarified by centrifugation (40,000 × g for 30 min). The supernatant was added to concanavalin A-Sepharose (400 μl of settled beads, equilibrated with Buffer A plus 0.1% DM) and incubated overnight at 4 °C while mixing. Washing occurred batchwise: the beads were pelleted using a clinical centrifuge (2,000 rpm for 3 min), the supernatant was removed, fresh buffer was added, and the beads were mixed for 10 min (4 °C). The beads were washed three times with 15 ml of Buffer A plus 0.1% DM, three times with Buffer B plus 0.1% DM, and two times with Buffer C plus 0.05% DM. After transferring the beads to a microcolumn, Rho was eluted with Buffer C plus 0.05% DM and 0.3 m methyl α-d-mannopyranoside. Rho concentration was ascertained by absorbance at 500 nm (ϵ = 40,800 liters cm-1 mol-1), and aliquots were snap frozen and stored at -80 °C. After thawing, Rho samples were centrifuged at 100,000 × g for 20 min, and the concentration was reassessed before use in experiments. Construction, Expression, and Purification of Arrestin—The bovine visual arrestin cDNA with a single glycine inserted at residue 2 (a generous gift from V. V. Gurevich) was cloned in the pET15b vector (Invitrogen) for bacterial expression. Mutant constructs W194F and I72C/W194F were created using PCR, and the constructs were verified by DNA sequencing. Arrestin was expressed in Escherichia coli BL21(DE3) cells and purified as described previously with some modifications (11Gurevich V.V. Benovic J.L. Methods Enzymol. 2000; 315: 422-437Crossref PubMed Google Scholar, 12Schubert C. Hirsch J.A. Gurevich V.V. Engelman D.M. Sigler P.B. Fleming K.G. J. Biol. Chem. 1999; 274: 21186-21190Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). A single colony was used to inoculate 400 ml of LB plus ampicillin (100 μg/ml), and this culture was grown while shaking at 37 °C overnight. This culture was then split between four flasks (each containing 1 liter of LB + ampicillin) and grown at 30 °C while shaking. Upon reaching an A595 of 0.6, the cultures were induced with 30 μm isopropyl 1-thio-β-d-galactopyranoside and grown for an additional 16–20 h. Cells were harvested by centrifugation (6,000 × g for 15 min), resuspended in cold Buffer D, and lysed by two passes through a French press (20,000 p.s.i.). The lysate was cleared by centrifugation (27,000 × g for 30 min). Ammonium sulfate was added to a concentration of 0.32 g/ml, and the precipitated protein was collected by centrifugation (27,000 × g for 30 min). The pellet was resuspended in Buffer E and centrifuged again before being dialyzed overnight against Buffer E plus 0.1 m NaCl. The dialyzed lysate was loaded onto a HiTrap heparin column (20 ml) equilibrated with Buffer E plus 0.1 m NaCl. The column was washed with ∼200 ml of Buffer E plus 0.1 m NaCl. After elution by a linear gradient of 0.1-0.5 m NaCl, the arrestin-containing fractions were determined by SDS-PAGE, pooled, and dialyzed overnight against Buffer F plus 0.1 m NaCl. The dialyzed fractions were loaded onto a HiTrap Q column (5 ml) equilibrated with Buffer F. The dialyzed fractions were diluted 1:10 with buffer F while loading onto the column. The loaded column was washed with 50 ml of Buffer F, and arrestin was eluted with a two-step gradient: 0-0.1 m and 0.1-0.5 m NaCl. The arrestin-containing fractions were pooled and concentrated to ∼2.5 mg/ml, snap frozen, and stored at -80 °C. The purity of the recombinant arrestin was >95% as ascertained by SDS-PAGE, and the yield was typically 5–6 mg of arrestin. Labeling of Arrestin—Arrestin samples were labeled with monobromobimane as described previously (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) except that no His tag selection was used. Briefly arrestin samples were buffer-exchanged (5 mm MES, 150 mm NaCl, pH 6.5) and concentrated, and monobromobimane was added in 10-fold molar excess to arrestin. After a 3-h incubation at room temperature, the majority of the free label was removed by ultrafiltration (Millipore Biomax). The labeled arrestin was then passed over a size exclusion column (500 μl, Sephadex G-15) to remove trace free label and buffer-exchanged into 20 mm HEPES, 150 mm NaCl, pH 7.4. The labeling efficiency was calculated as described previously (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar); recombinant arrestin I72C/W194F labeled at ∼92% efficiency, and recombinant arrestin W194F labeled at less than 2% efficiency. No free label contamination was detected in the labeled arrestin samples. Centrifugal pull-down analysis (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) showed arrestin W194F and bimane-labeled I72C/W194F to have essentially the same affinity for Rho*-P as that of wild-type arrestin (data not shown). Preparation of Mixed Micelles—For experiments using mixed micelles of ROS phospholipids and DM, ROS containing 50 μm Rho-P was solubilized in 1% DM. The membranes were dispersed in the detergent by continuous sonication (25 °C for 2 min, Branson 1210) followed by centrifugation at 100,000 × g for 15 min to pellet the insoluble material. This stock of solubilized ROS-P was diluted into buffer containing different amounts of DM, and samples were sonicated briefly and incubated at 25 °C for 1 h to allow equilibration of the micelles. For experiments using asolectin, an appropriate volume of 1% DM was added to a portion of powdered asolectin to yield a stock of 1% asolectin/DM. The solution was passed multiple times through a fine needle to disperse the phospholipids and was clarified by centrifugation (100,000 × g for 20 min) before use. For all experiments, DM and asolectin stocks were diluted in 20 mm HEPES, 150 mm NaCl, pH 7.4, to give a final DM concentration of 0.02%. For molarity calculations of asolectin, an average phospholipid molecular mass of 750 g/mol was assumed. For experiments using purified phospholipids, a measured volume of the chloroform stock corresponding to 1 mg of phospholipid (as supplied from Avanti) was dispersed into a glass test tube, and the chloroform was evaporated with a continuous stream of argon. The dried lipid film was then resuspended in a volume of 1% DM in buffer (20 mm HEPES, 150 mm NaCl, pH 7.4) by vortexing and multiple freeze-thaw cycles to give a final phospholipid concentration of 5 mm. All phospholipid suspensions were stored in the dark and handled under argon to avoid lipid oxidation, and lipid stocks were clarified by centrifugation (30,000 × g for 10 min) before use. Fluorescence Spectroscopy—All steady-state and time-resolved fluorescence measurements were made as described previously (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The tryptophanless arrestin mutant W194F was used, for it contributes less background while measuring opsin tryptophan fluorescence in samples of Rho and arrestin. Because asolectin exhibits some intrinsic fluorescence, background controls were measured and subtracted from appropriate fluorescence spectra. UV-visible Absorbance Spectroscopy—All UV-visible absorption spectra were recorded with a Shimadzu UV-1601 spectrophotometer using a bandwidth of 2 nm. For the photodecay experiments, the absorbance of 1 μm Rho-P (120 μl) was recorded in the dark after base lining with the appropriate buffer. The sample was photoactivated using a 150-watt fiber optic light source (>495 nm) for 20 s, and spectra were subsequently recorded every 90 s for 120 min. The presence of Schiff base was ascertained by the addition of 5 μl of 0.8 n H2SO4. NaBH4 Reduction and V8 Proteolysis of Rho—Reduction of the Schiff base in Rho with NaBH4 results in the fluorescent n-retinylidene opsin species (λex, 340 nm; λem, 480 nm) (13Bownds D. Wald G. Nature. 1965; 205: 254-257Crossref PubMed Scopus (101) Google Scholar, 14Farrens D.L. Khorana H.G. J. Biol. Chem. 1995; 270: 5073-5076Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Samples of 3 μm Rho-P, with or without 6 μm arrestin, solubilized in 0.02% DM, 0.02% DM and 0.02% asolectin, or 0.02% DM and 100 μm purified phospholipid (20 μl) were photoactivated using a 150-watt fiber optic light source (>495 nm) for 20 s and allowed to decay at 20 °C in the dark. After 120 min, 5 μl of 1% NaBH4 (made fresh in water) was added to each sample. After 10 min, 15 μl of 1 m sodium phosphate (pH 7.0) was added, each sample was split into two 20-μl aliquots, and 5 μl of 4.8 μm V8 protease was added to half the samples. Proteolysis occurred for 30 min at room temperature. To assess the amount of Schiff base present in Meta II Rho immediately after activation, 1% NaBH4 was added to Rho-P (0.02% DM) in the dark. The sample was then photoactivated at 4 °C and immediately processed as described above. Bands were resolved by 15% Tris-Tricine SDS-PAGE, and gels were soaked in 30% methanol before visualization. The n-retinylidene opsin was excited with a short wave UV source (Alpha-Innotech FluorChem 5500 imaging system), and the fluorescent bands were detected by a charge-coupled device camera (535 ± 50-nm cutoff filter, 10-min exposure). AlphaEase FC software was used to quantify the fluorescence of the bands. Our results here indicate the following. 1) Phospholipids are required to enable Rho-arrestin interactions in detergent micelles. 2) In mixed DM/phospholipid micelles, arrestin interacts with Meta III and converts it to a Meta II-like species. 3) Arrestin release is significantly inhibited from Rho*-P in mixed micelles containing acidic phospholipids, while half of the retinal is trapped in the binding pocket. Details are given below. Arrestin Has Reduced Affinity for DM-purified Rho*-P—The binding of bimane-labeled arrestin I72C/W914F (I72B) to Rho*-P in native membranes resulted in an increase (∼40%) and a blue shift (∼15 nm) in fluorescence (Fig. 2A). However, when Rho-P was purified from ROS and solubilized in DM, the fluorescence changes were dramatically reduced, indicating a loss of arrestin binding (Fig. 2B). Asolectin Stimulates Arrestin Binding to DM-purified Rho*-P—Adding asolectin (a mixture of phospholipids often used in Rho reconstitution (15Niu L. Kim J.M. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13409-13412Crossref PubMed Scopus (28) Google Scholar)), to DM-purified Rho-P restores I72B binding: the fluorescence increased ∼70% and blue shifted ∼15 nm after photoactivation (Fig. 2C). Dilution of ROS Lipids with Detergent Inhibits Arrestin Binding to Rho*-P—We determined the effect of decreasing the apparent concentration of native ROS lipids on arrestin binding using DM (Fig. 2D). Rather than purify ROS lipids and add them back to purified Rho-P, we fully solubilized native ROS-P membranes and then added this mixture to buffer containing low to high concentrations of DM (0.02–1.0%). In this way, the relative Rho/phospholipid ratio was preserved, but the number of micelles into which the phospholipids could segregate was increased. This process ensured that minimal oxidation or damage occurred to the ROS lipids. A similar approach has been used previously in control experiments involving ROS lipids (16Gibson N.J. Brown M.F. Biochemistry. 1993; 32: 2438-2454Crossref PubMed Scopus (163) Google Scholar). When this DM-solubilized ROS-P was added to 0.02% DM (micelle concentration, ∼3 μm), 3This calculation assumes that the aggregation number of 132 molecules of DM/micelle (57Dupuy C. Auvray X. Petipas C. Langmuir. 1997; 13: 3965Crossref Scopus (105) Google Scholar) is not dramatically changed by the presence of ROS phospholipids. arrestin could bind as indicated by the ∼70% increase and 15-nm blue shift in fluorescence. However, at higher DM concentrations, binding was dramatically inhibited (Fig. 2D). In 1% DM (micelle concentration, ∼150 μm), the fluorescence of arrestin I72B increased only ∼17% and blue shifted less than 2 nm after light activation. Assuming the ROS phospholipids distribute evenly to all micelles, we interpret this result to reflect the ability of DM to solubilize ROS phospholipids away from Rho. Although DM might directly inhibit arrestin binding, the addition of sufficient amounts of exogenous phospholipids, even at high DM concentrations, could rescue arrestin binding (see Fig. 2E, inset). ∼50 Phospholipids per Rho-P Are Required for Arrestin Binding—We quantified the phospholipid effect by titrating asolectin in samples of DM-purified Rho-P (Fig. 2E). These studies indicate that ∼0.015% asolectin, or ∼200 μm phospholipid, is required to achieve maximal arrestin binding under conditions where there is roughly one Rho per micelle. This corresponds to ∼67 phospholipids per Rho. Interestingly this is similar to the Rho/phospholipid ratio in the ROS where Rho composes half the volume of the tightly stacked membranous organelles (17Anderson R.E. Maude M.B. Biochemistry. 1970; 9: 3624-3628Crossref PubMed Scopus (163) Google Scholar, 18Daemen F.J. Biochim. Biophys. Acta. 1973; 300: 255-288Crossref PubMed Scopus (266) Google Scholar, 19Molday R.S. Investig. Ophthalmol. Vis. Sci. 1998; 39: 2491-2513PubMed Google Scholar). When 10-fold more DM was present, correspondingly more phospholipid was required (0.1–0.12% asolectin) (Fig. 2E, inset). Again this value corresponds to ∼50 phospholipids per micelle. Some Retinal and Arrestin Release Is Inhibited in Mixed Micelles—We used a fluorescence dual rate assay (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) to investigate the various dynamics of arrestin and retinal release. This assay monitors release of retinal as an increase in the tryptophan fluorescence of opsin (14Farrens D.L. Khorana H.G. J. Biol. Chem. 1995; 270: 5073-5076Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) while simultaneously measuring dissociation of arrestin I72B as a decrease in bimane fluorescence. We determined the maximal and minimal fluorescence values possible for each process (the “plateaus”) by adding hydroxylamine, a compound that cleaves the Schiff base and converts all remaining photoproducts to opsin and free retinaloxime. Fig. 3, A, C, and E, compare retinal and arrestin release from Rho*-P in pure DM micelles or mixed micelles containing ROS phospholipids or asolectin. The half-lives (t½) of retinal and arrestin release and the relative levels of retinal trapping and arrestin binding are given in Tables 1 and 2. The data are briefly summarized below.TABLE 1Effect of arrestin on the rate of retinal release and amount of trapped retinal Values are derived from experiments described in Figs. 3 and 5 and, for the native membrane sample, from work published previously (5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Values represent the average ± S.E. from two independent experiments (20 °C, pH 7.4).Membrane or micelleNo arrestin+ arrestint½ retinal releaseaSingle exponential rates were measured and determined as described under “Experimental Procedures” and converted to t½ values (t½ = ln2/k where k is the rate constant in min–1). In each experiment, 1 μm Rho-P ± 2 μm arrestin I72B were used except for the native membrane sample where 2 μm Rho-P ± 4 μm arrestin were usedPercentage of retinal trappedbThe percentage of trapped retinal was calculated from the Rho-P tryptophan fluorescence retinal release data (330 nm) as follows: [(c – a)/c] × 100 where a is the (F/F0) – 1 value before the addition of NH2OH, and c is the (F/F0) – 1 value after the addition of NH2OH (see Fig. 6A for more details)t½ retinal releaseaSingle exponential rates were measured and determined as described under “Experimental Procedures” and converted to t½ values (t½ = ln2/k where k is the rate constant in min–1). In each experiment, 1 μm Rho-P ± 2 μm arrestin I72B were used except for the native membrane sample where 2 μm Rho-P ± 4 μm arrestin were usedPercentage of retinal trappedbThe percentage of trapped retinal was calculated from the Rho-P tryptophan fluorescence retinal release data (330 nm) as follows: [(c – a)/c] × 100 where a is the (F/F0) – 1 value before the addition of NH2OH, and c is the (F/F0) – 1 value after the addition of NH2OH (see Fig. 6A for more details)minminNative membranes7.5 ± 0.3NDcND, not determined because of secondary fluorescence effects observed to occur when NH2OH is added to native membranes8.9 ± 0.7NDNative membranes solubilized by DM14.9 ± 0.55 ± 122.5 ± 0.311 ± 1DM micelles7.9 ± 0.319 ± 18.6 ± 0.220 ± 2DM/asolectin micelles12.3 ± 0.85 ± 013.2 ± 0.752 ± 4DM/PC12.0 ± 0.914 ± 214.0 ± 0.216 ± 1DM/PE12.4 ± 0.81 ± 014.0 ± 0.33 ± 1DM/PS12.6 ± 1.16 ± 312.8 ± 0.358 ± 4DM/PI13.2 ± 1.417 ± 313.6 ± 0.149 ± 1DM/PA8.8 ± 0.65 ± 111.1 ± 0.660 ± 3a Single exponential rates were measured and determined as described under “Experimental Procedures” and converted to t½ values (t½ = ln2/k where k is the rate constant in min–1). In each experiment, 1 μm Rho-P ± 2 μm arrestin I72B were used except for the native membrane sample where 2 μm Rho-P ± 4 μm arrestin were usedb The percentage of trapped retinal was calculated from the Rho-P tryptophan fluorescence retinal release data (330 nm) as follows: [(c – a)/c] × 100 where a is the (F/F0) – 1 value before the addition of NH2OH, and c is the (F/F0) – 1 value after the addition of NH2OH (see Fig. 6A for more details)c ND, not determined because of secondary fluorescence effects observed to occur when NH2OH is added to native membranes Open table in a new tab TABLE 2Arrestin I72B fluorescence changes due to Rho*-P binding and release Values are derived from experiments described in Figs. 3 and 5 and, for the native membrane sample, from Ref. 5Sommer M.E. Smith W.C. Farrens D.L. J. Biol. Chem. 2005; 280: 6861-6871Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar and M. E. Sommer, W. C. Smith, and D. L. Farrens (unpublished work). Values represent the average ± S.E. from two independent experiments (20 °C, pH 7.4).Membrane or micelle(F/F0) – 1 (456 nm)Percentage of residual fluorescenceaDetermined from the average fluorescence intensity of arrestin I72B 120 min after light activation (or 200 min for DM-solubilized ROS-P) divided by the average intensity immediately after light activationt½ of arrestin releasebSingle exponential rates were measured and determined as described under “Experimental Procedures” and converted to t½ values (t½ = ln2/k where k is the rate constant in min–1). In each experiment, 1 μm Rho-P and 2 μm arrestin I72B were used except for the native membrane sample where 2 μm Rho-P and 4 μm arrestin were used. Rates of arrestin release were measured during the same experiment in which retinal release was measured for Table 1Arrestin bindingArrestin plateauminNative membranes0.42 ± 0.08cThe fluorescence intensity of arrestin I72B in the presence of native membranes is complicated by the variable amount of scatter generated by different membrane preparations0.09 ± 0.02cThe fluorescence intensity of arrestin I72B in the presence of native membranes is complicated by the variable amount of scatter generated by different membrane preparations219.4 ± 1.1Native membranes solubilized by DM0.5 ± 0.10.1 ± 0.012028.0 ± 0.7DM micelles0.12 ± 0.10.03 ± 0.02258.1 ± 0.4DM/asolectin micelles0.5 ± 0.20.25 ± 0.025023.4 ± 0.4DM/PC0.28 ± 0.20.06 ± 0.012119.5 ± 1.3DM/PE0.24 ± 0.10.07 ± 0.012919.8 ± 0.6DM/PS0.68 ± 0.10.62 ± 0.0491>120DM/PI0.62 ± 0.10.55 ± 0.0189>120DM/PA0.93 ± 0.10.87 ± 0.0793>120a Determined from the average fluorescence intensity of arrestin I72B 120 min after light activation (or 200 min for DM-solubilized ROS-P) divided by the average intensity immediately after light activationb Single exponential rates were measured and determined as described under “Experimental Procedures” and converted to t½ values (t½ = ln2/k where k is the rate constant in min–1). In each experiment, 1 μm R" @default.
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• W2037594168 date "2006-04-01" @default.
• W2037594168 modified "2023-10-07" @default.
• W2037594168 title "Dynamics of Arrestin-Rhodopsin Interactions" @default.
• W2037594168 cites W1506213416 @default.
• W2037594168 cites W193979248 @default.
• W2037594168 cites W1966466658 @default.
• W2037594168 cites W1967326841 @default.
• W2037594168 cites W1969306961 @default.
• W2037594168 cites W1970524455 @default.
• W2037594168 cites W1974597502 @default.
• W2037594168 cites W1983653244 @default.
• W2037594168 cites W1985540529 @default.
• W2037594168 cites W1987512475 @default.
• W2037594168 cites W1991869401 @default.
• W2037594168 cites W1994019984 @default.
• W2037594168 cites W1996783056 @default.
• W2037594168 cites W2007512612 @default.
• W2037594168 cites W2008045893 @default.
• W2037594168 cites W2018966173 @default.
• W2037594168 cites W2021312727 @default.
• W2037594168 cites W2027825638 @default.
• W2037594168 cites W2029321566 @default.
• W2037594168 cites W2029928876 @default.
• W2037594168 cites W2030222651 @default.
• W2037594168 cites W2034782869 @default.
• W2037594168 cites W2041200489 @default.
• W2037594168 cites W2044627868 @default.
• W2037594168 cites W2046562640 @default.
• W2037594168 cites W2052288973 @default.
• W2037594168 cites W2052792449 @default.
• W2037594168 cites W2054308102 @default.
• W2037594168 cites W2054508286 @default.
• W2037594168 cites W2061615383 @default.
• W2037594168 cites W2063413026 @default.
• W2037594168 cites W2064382411 @default.
• W2037594168 cites W2066299155 @default.
• W2037594168 cites W2066307488 @default.
• W2037594168 cites W2067990965 @default.
• W2037594168 cites W2068067779 @default.
• W2037594168 cites W2069867836 @default.
• W2037594168 cites W2072073925 @default.
• W2037594168 cites W2072216679 @default.
• W2037594168 cites W2072771599 @default.
• W2037594168 cites W2076770170 @default.
• W2037594168 cites W2085471204 @default.
• W2037594168 cites W2098449500 @default.
• W2037594168 cites W2100027801 @default.
• W2037594168 cites W2116880855 @default.
• W2037594168 cites W2118950099 @default.
• W2037594168 cites W2123888304 @default.
• W2037594168 cites W2130049582 @default.
• W2037594168 cites W2136346557 @default.
• W2037594168 cites W2140704050 @default.
• W2037594168 cites W2152339698 @default.
• W2037594168 cites W2155909615 @default.
• W2037594168 cites W2169217477 @default.
• W2037594168 cites W2411368169 @default.
• W2037594168 doi "https://doi.org/10.1074/jbc.m510037200" @default.
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