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• W2005661568 abstract "Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (Ea) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of ∼2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories. Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (Ea) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of ∼2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories. Rhodopsin, the dim light photoreceptor of rod cells, is arguably the best characterized member of the class A superfamily of GPCR (1Ebrey T. Koutalos Y. Prog. Retin. Eye Res. 2001; 20: 49-94Crossref PubMed Scopus (347) Google Scholar, 2Hubbell W.L. Altenbach C. Hubbell C.M. Khorana H.G. Adv. Protein Chem. 2003; 63: 243-290Crossref PubMed Scopus (339) Google Scholar, 3Meng E.C. Bourne H.R. Trends Pharmacol. Sci. 2001; 22: 587-593Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 4Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 5Sakmar T.P. Menon S.T. Marin E.P. Awad E.S. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 443-484Crossref PubMed Scopus (202) Google Scholar, 6Ridge K.D. Abdulaev N.G. Sousa M. Palczewski K. Trends Biochem. Sci. 2003; 28: 479-487Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 7Filipek S. Stenkamp R.E. Teller D.C. Palczewski K. Annu. Rev. Physiol. 2002; 20: 20Google Scholar). A transmembrane receptor, it has evolved into an efficient photoreceptor by covalently binding its chromophore, 11-cis-retinal, to lysine 296 via a protonated Schiff base linkage within the helical bundle (8Wald G. Science. 1968; 162: 230-239Crossref PubMed Scopus (785) Google Scholar, 9Palczewski K. Kumasaka T. Hori T. Behnke C.A. Motoshima H. Fox B.A. Le Trong I. Teller D.C. Okada T. Stenkamp R.E. Yamamoto M. Miyano M. Science. 2000; 289: 739-745Crossref PubMed Scopus (4991) Google Scholar). Dim light vision begins when the 11-cis-retinal chromophore absorbs a photon and isomerizes to the all-trans-retinal form. This change in retinal initiates a series of photo-intermediates and conformational changes in the protein, resulting in the formation of metarhodopsin II (MII), 1The abbreviations used are: MII, metarhodopsin II; DM, n-dodecyl β-d-maltoside; MES, 2-(N-morpholino)ethanesulfonic acid; Ea, energy of activation; GTPγS, guanosine 5′-3-O-(thio)triphosphate; WT, wild-type; λmax, absorbance maxima.1The abbreviations used are: MII, metarhodopsin II; DM, n-dodecyl β-d-maltoside; MES, 2-(N-morpholino)ethanesulfonic acid; Ea, energy of activation; GTPγS, guanosine 5′-3-O-(thio)triphosphate; WT, wild-type; λmax, absorbance maxima. the active conformation of rhodopsin that is able to bind and activate the G-protein transducin. The MII photoproduct is in dynamic equilibrium with its predecessor MI, and this MI/MII pool is thought to decay through two processes (10Vogel R. Siebert F. Mathias G. Tavan P. Fan G. Sheves M. Biochemistry. 2003; 42: 9863-9874Crossref PubMed Scopus (44) Google Scholar). The MII product may be directly hydrolyzed and release all-trans-retinal from the binding pocket, or the MI pool may undergo an addition thermal isomerization along the chromophore C=N double bond (all-trans 15-syn) giving rise to the MIII storage product (10Vogel R. Siebert F. Mathias G. Tavan P. Fan G. Sheves M. Biochemistry. 2003; 42: 9863-9874Crossref PubMed Scopus (44) Google Scholar). This MIII intermediate also decays to opsin and all-trans-retinal (albeit at a slower rate) either through the MI/MII pool or possibly direct retinal Schiff base hydrolysis of the MIII intermediate.Rhodopsin deactivation ultimately requires hydrolysis of the all-trans-retinal Schiff base linkage and release of retinal from the binding pocket. Recycling the receptor and returning it to a photosensitive conformational state completes the recovery process (11Palczewski K. Saari J.C. Curr. Opin. Neurobiol. 1997; 7: 500-504Crossref PubMed Scopus (69) Google Scholar). The retinoid cycle accomplishes this task by converting the released all-trans-retinal back to the 11-cis conformation through a series of enzymatic reactions, eventually resulting in the reformation of the retinal Schiff base linkage and regeneration of the photosensitive receptor (12McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin. Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (314) Google Scholar, 13Rando R.R. Chem. Rev. 2001; 101: 1881-1896Crossref PubMed Scopus (139) Google Scholar). Although extensive research into Schiff base formation and the retinoid cycle has resulted in a wealth of knowledge (12McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin. Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (314) Google Scholar, 13Rando R.R. Chem. Rev. 2001; 101: 1881-1896Crossref PubMed Scopus (139) Google Scholar), little is known about the molecular mechanism of Schiff base hydrolysis and the subsequent retinal release both in the dark state or during the decay of the functional MII state (12McBee J.K. Palczewski K. Baehr W. Pepperberg D.R. Prog. Retin. Eye Res. 2001; 20: 469-529Crossref PubMed Scopus (314) Google Scholar, 14Baehr W. Wu S.M. Bird A.C. Palczewski K. Vision Res. 2003; 43: 2957-2958Crossref PubMed Scopus (50) Google Scholar).Although both rod and cone opsins bind their retinal chromophores through a Schiff base linkage (1Ebrey T. Koutalos Y. Prog. Retin. Eye Res. 2001; 20: 49-94Crossref PubMed Scopus (347) Google Scholar, 15Stenkamp R.E. Filipek S. Driessen C.A. Teller D.C. Palczewski K. Biochim. Biophys. Acta. 2002; 1565: 168-182Crossref PubMed Scopus (96) Google Scholar), this linkage is markedly less stable in cone opsins, and as a result the turnover and regeneration rates for cone cells are significantly faster than for rod cells (16Shichida Y. Imai H. Imamoto Y. Fukada Y. Yoshizawa T. Biochemistry. 1994; 33: 9040-9044Crossref PubMed Scopus (72) Google Scholar, 17Imai H. Kojima D. Oura T. Tachibanaki S. Terakita A. Shichida Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2322-2326Crossref PubMed Scopus (119) Google Scholar, 18Babu K.R. Dukkipati A. Birge R.R. Knox B.E. Biochemistry. 2001; 40: 13760-13766Crossref PubMed Scopus (47) Google Scholar, 19Starace D.M. Knox B.E. J. Biol. Chem. 1997; 272: 1095-1100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Furthermore, although the retinal Schiff base linkage is quite stable in dark state rhodopsin, it hydrolyzes quickly in the active MII signaling state of the protein (1Ebrey T. Koutalos Y. Prog. Retin. Eye Res. 2001; 20: 49-94Crossref PubMed Scopus (347) Google Scholar, 20Cooper A. Dixon S. Nutley M. Robb J. J. Am. Chem. Soc. 1987; 109: 7254-7263Crossref Scopus (30) Google Scholar, 21Steinberg G. Ottolenghi M. Sheves M. Biophys. J. 1993; 64: 1499-1502Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 22Doukas A.G. Aton B. Callender R.H. Ebrey T.G. Biochemistry. 1978; 17: 2430-2435Crossref PubMed Scopus (124) Google Scholar), suggesting that some change probably occurs in the vicinity of the Schiff base attachment site to account for this disparity. Clearly the apoprotein plays a significant role in stabilizing the retinal Schiff base linkage.Recently, a high resolution structure of rhodopsin suggested the presence of a hydrogen bond network encompassing the retinal Schiff base attachment site near extracellular loop E-2 and the “retinal plug” domain (23Okada T. Fujiyoshi Y. Silow M. Navarro J. Landau E.M. Shichida Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5982-5987Crossref PubMed Scopus (650) Google Scholar). This network consists of both the carbonyl backbone and side chains of amino acids lining the retinal binding pocket as well as water molecules within the pocket that surround the retinal Schiff base linkage. This network was recently demonstrated to play a key role in the protonated Schiff base counter ion switch mechanism proposed to occur upon formation of the MI photointermediate subsequent to light activation (24Yan E.C. Kazmi M.A. Ganim Z. Hou J.M. Pan D. Chang B.S. Sakmar T.P. Mathies R.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9262-9267Crossref PubMed Scopus (182) Google Scholar). However, the role that this retinal hydrogen bond network plays in stabilizing the retinal Schiff base and in potentially participating in the mechanism of Schiff base hydrolysis remains as yet unexplored.In the this study we report our investigation into the role of the retinal hydrogen bond network in relation to the retinal Schiff base linkage in rhodopsin. Through site-directed mutagenesis, we find that disruption of this network results in compromised stability of the Schiff base linkage in the dark state and a loss of concave Arrhenius plots for all of the mutant proteins (especially for residues Thr94 and Glu113). In addition, we find that mutation of this network also affects Schiff base stability in the MII state, but only at some sites. Paradoxically, mutation of these sites (Thr94, Glu113 and Ser186) actually slows MII decay, suggesting that these residues act as participants in Schiff base hydrolysis and retinal release process. Finally, through deuterium isotope studies, we provide evidence that multiple proton transfer events occur during the process of Schiff base hydrolysis and subsequent retinal release and that this process proceeds through a carbinolamine intermediate. Our results further illustrate how the rhodopsin structure maintains Schiff base integrity and provides insight to the mechanism of Schiff base hydrolysis and retinal release.EXPERIMENTAL PROCEDURESMaterials and Buffers—Except where noted, all buffers and chemicals were purchased from either Fisher or Sigma. n-Dodecyl β-d-maltoside (DM) was purchased from Anatrace (Maumee, OH), Polystyrene columns (2-ml bed volume) were purchased from Pierce. Frozen bovine retinas were from J. A. Lawson Co. (Lincoln, NE). Transducin was purified from rod outer segments as described previously (25Baehr W. Morita E.A. Swanson R.J. Applebury M.L. J. Biol. Chem. 1982; 257: 6452-6460Abstract Full Text PDF PubMed Google Scholar). Restriction endonucleases were from New England Biolabs (Beverly, MA). 11-cis-Retinal was a generous gift from Dr. R. Crouch (Medical University of South Carolina and the National Eye Institute). The 1D4 antibody was purchased from the National Cell Culture Center (Minneapolis, MN). The nonapeptide corresponding to the C terminus of rhodopsin was acquired from the Emory University Microchemical Facility (Atlanta, GA). Cuvettes were purchased from Uvonics (Plainview, NY). Deuterium oxide was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). Centrifugal filter devices (0.5 μm volume, 10-kDa cut off) were purchased from Millipore (Billerica, MA). Definitions for the buffers used are as follows: PBSSC (0.137 M NaCl, 2.7 mm KCl, 1.5 mm KH2PO4, and 8 mm Na2HPO4 (pH 7.2)), buffer A (1% DM and PBSSC (pH 7.2)), buffer B (2 mm ATP, 0.1% DM, 1 m NaCl, and 2 mm MgCl2 (pH 7.2)), buffer C (0.05% DM and PBSSC (pH 7.0)), buffer D (0.05% DM and 5 mm MES (pH 6.0)).Construction, Expression, and Purification of Rhodopsin Mutants—Site-directed mutagenesis was performed using overlap extension PCR to generate fragments containing the mutation of interest (T94I, E113Q, E181Q, S186A, Y192F, and Y268F) in the synthetic bovine rhodopsin gene (26Ferretti L. Karnik S.S. Khorana H.G. Nassal M. Oprian D.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 599-603Crossref PubMed Scopus (160) Google Scholar) essentially as described previously and subcloned into the pMT4 plasmid (27Janz J.M. Farrens D.L. Biochemistry. 2001; 40: 7219-7227Crossref PubMed Scopus (42) Google Scholar, 28Janz J.M. Fay J.F. Farrens D.L. J. Biol. Chem. 2003; 278: 16982-16991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) for expression. All mutations were confirmed by the dideoxynucleotide sequencing method.The mutant rhodopsin proteins were transiently expressed in COS-1 cells using the DEAE-dextran method, and cells were harvested 56-72 h after transfection as described previously (27Janz J.M. Farrens D.L. Biochemistry. 2001; 40: 7219-7227Crossref PubMed Scopus (42) Google Scholar, 29Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (382) Google Scholar). The harvested opsin mutants were subsequently regenerated with 10 μm 11-cis-retinal at 4 °C for 1 h followed by an additional 5 μm of 11-cis retinal and 1-h incubation (30Reeves P.J. Hwa J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1927-1931Crossref PubMed Scopus (52) Google Scholar). The purification of the rhodopsin mutants proceeded essentially as the original procedure (29Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (382) Google Scholar) with modifications as previously described (28Janz J.M. Fay J.F. Farrens D.L. J. Biol. Chem. 2003; 278: 16982-16991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 31Dunham T.D. Farrens D.L. J. Biol. Chem. 1999; 274: 1683-1690Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). A spectrum of each elution fraction was recorded, and the purified samples were either used immediately or snap frozen in liquid N2 and stored at -80 °C.UV-visible Absorption Spectroscopy—All UV-visible absorption spectra were recorded with a Shimadzu UV-1601 spectrophotometer at 20 °C using a bandwidth of 2 nm, a response time of 1 s, and a scan speed of 500 nm/min unless otherwise noted. For concentration calculations, the molar extinction coefficient value (ϵ500) for WT rhodopsin was taken to be 40 600 m-1 cm-1 (32Wald G. Brown P.K. J. Gen. Physiol. 1953; 37: 189-200Crossref PubMed Scopus (288) Google Scholar). The samples were photobleached in buffer A by illumination for 30 s (at a 6-Hz flash rate) with a Machine Vision Strobe light source (EG & G) equipped with a wavelength >490 nm long pass filter. This light treatment was found to be adequate for full conversion of all samples. The presence of a protonated Schiff base in the MII state for each mutant was verified by adding H2SO4 to a pH of 1.9 immediately following photobleaching then measuring the absorbance spectrum to assay the presence of a spectral species at 440 nm (which indicates an intact retinal protonated Schiff base) (33Sakamoto T. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 249-253Crossref PubMed Scopus (34) Google Scholar). Photobleaching time course analysis on mutant Y268F was performed as above but with bleaching time reduced to 15 s, and results compared with a WT rhodopsin irradiated in the exact same manner.Determination of Transducin (GT) Activation Rates—Activation of GT by rhodopsin was monitored using fluorescence spectroscopy at 10 °C as described previously (34Phillips W.J. Cerione R.A. J. Biol. Chem. 1988; 263: 15498-15505Abstract Full Text PDF PubMed Google Scholar, 35Fahmy K. Sakmar T.P. Biochemistry. 1993; 32: 7229-7236Crossref PubMed Scopus (133) Google Scholar, 36Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1103) Google Scholar) using a Photon Technologies QM-1 steady state fluorescence spectrophotometer (37Farrens D.L. Khorana H.G. J. Biol. Chem. 1995; 270: 5073-5076Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). The excitation wavelength was 295 nm (2-nm bandwidth), and fluorescence emission was monitored at 340 nm (12-nm bandwidth). Briefly, photobleached mutant rhodopsin (final concentration of 5 nm) was added to the reaction mixture consisting of 250 nmGT in 10 mm Tris (pH 7.2), 2 mm MgCl2, 100 mm NaCl, 1 mm dithiothreitol, and 0.01% DM. Thus the mixture was allowed to stir for 300 s; then the reaction was initiated by the addition of GTPγS to a final concentration of 5 μm, and the increase in fluorescence was followed for an additional 1500 s. To calculate the activation rates, the slopes of the initial fluorescence increase following GTPγS addition were determined through the data points covering the first 60 s using linear regression analysis. Mutant rhodopsin activation values were reported as percentages relative to WT, which was taken to be 100%.Thermal Bleaching of Rhodopsin Samples—Thermal decay rates were followed by UV-visible spectroscopy in buffer A essentially as described previously (28Janz J.M. Fay J.F. Farrens D.L. J. Biol. Chem. 2003; 278: 16982-16991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 38Janz J.M. Farrens D.L. Vision Res. 2003; 43: 2991-3002Crossref PubMed Scopus (31) Google Scholar). The thermal stability of each mutant was determined by first monitoring the absorbance of each sample from 650-250 nm at 1-min intervals at a given temperature. Thermal decay rates were subsequently obtained by monitoring the decrease of the 500 nm absorbing dark state species from these measurements over time (39Davidson F.F. Loewen P.C. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4029-4033Crossref PubMed Scopus (129) Google Scholar, 40Andres A. Kosoy A. Garriga P. Manyosa J. Eur. J. Biochem. 2001; 268: 5696-5704Crossref PubMed Scopus (25) Google Scholar, 41Vogel R. Siebert F. Biochemistry. 2002; 41: 3529-3535Crossref PubMed Scopus (27) Google Scholar). Baseline drift was corrected for by normalizing all spectra to an absorbance of zero at 650 nm. For E113Q, thermal decay rates were measured by monitoring the increase in tryptophan fluorescence at 330 nm, which was caused by the release of retinal from the chromophore-binding pocket (37Farrens D.L. Khorana H.G. J. Biol. Chem. 1995; 270: 5073-5076Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) as described previously (28Janz J.M. Fay J.F. Farrens D.L. J. Biol. Chem. 2003; 278: 16982-16991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 38Janz J.M. Farrens D.L. Vision Res. 2003; 43: 2991-3002Crossref PubMed Scopus (31) Google Scholar). The experimental setup for these assays is similar to that of the retinal release assay (described below) except that the samples were not photobleached during the assay. All thermal decay data was analyzed using monoexponential decay (absorbance experiments) or monoexponential rise to maximum (fluorescence experiments) fitting algorithms in Sigma Plot (Jandel Scientific Software). Ea values for mutant thermal decay assays were determined by applying the rate data to the Arrhenius equation: k = Ae-Ea/(RT), as described previously (28Janz J.M. Fay J.F. Farrens D.L. J. Biol. Chem. 2003; 278: 16982-16991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar).Measurement of the Rate of Retinal Release and/or MII Decay by Fluorescence Spectroscopy—The MII stability was assessed by measuring the time course of retinal release coinciding with the rate of decay for the active MII state using the PTI fluorimeter described above. Each measurement was carried out using 100 μl of a 0.25 μm mutant sample in buffer A, and the sample temperature was maintained as described above. After the samples were photobleached to the MII state (see above), the retinal release measurements were carried out at the appropriate temperature by exciting the sample for 3 s (excitation wavelength = 295 nm, 1/4 nm bandwidth slit setting) and then blocking the excitation beam for 42 s to avoid further photobleaching the samples. Tryptophan fluorescence emission was monitored at 330 nm (12-nm bandwidth slit setting), and this cycle was repeated until a plateau in the signal was achieved. To determine the t12 values for retinal release, experimental data were analyzed using a monoexponential rise to maximum fit in Sigma Plot. In this manner series of MII decay rates were obtained at 5, 10, 15, 20, 25, 30, and 35 °C, and their rates were applied to the Arrhenius equation, k = Ae-Ea/(RT), to determine the Ea value of the retinal release process for each mutant rhodopsin.Hydroxylamine Reactivity—Hydroxylamine reactivity of the dark state was determined for purified WT and mutant rhodopsins by monitoring the rate of 500 nm absorbance decrease after the addition of hydroxylamine (pH 6.0) to the samples in buffer A to a final concentration of 50 mm at the indicated temperatures (28Janz J.M. Fay J.F. Farrens D.L. J. Biol. Chem. 2003; 278: 16982-16991Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 42Sakmar T.P. Franke R.R. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3079-3083Crossref PubMed Scopus (130) Google Scholar). Baseline drift was corrected as described above (see “Thermal Bleaching of Rhodopsin Samples”).Solvent Isotope Effects on Dark State Stability and MII Decay Rates—A 10% DM stock and a 500 mm MES (pH 6.0) stock buffer were made up fresh in deuterium oxide (D2O) and were then used to make a 0.05% DM, 5 mm MES (pH 6.0) (buffer D) D2O exchange buffer. The pH of the D2O experiments ranged between 6.0 and 6.4 because the pH for the deuterium buffer is expected to be off by + 0.4 pH units. However, as the process of MII decay has been shown to be independent over a pH range of 5.0-8.0 (38Janz J.M. Farrens D.L. Vision Res. 2003; 43: 2991-3002Crossref PubMed Scopus (31) Google Scholar), this slight pH change should not alter interpretation of the results. Using a concentrated stock of purified rhodopsin (to use a small volume for dilution) two equal molar stocks of both D2O and H2O rhodopsin were prepared. These samples (500 nm) were then buffer-exchanged using Millipore Ultrafree 0.5 centrifugal filter devices at low spin speeds. The D2O sample was washed eight times with 500 μl of D2O-containing buffer, whereas the H2O sample was washed in the exact same method using H2O buffer to ensure that any detergent concentration that may have taken place during the exchange was equal for both samples. The samples were then assayed for dark state thermal decay (55 and 37 °C) as well as MII decay rates at 20 °C as described above. The procedure was repeated and values were obtained from three separate experiments from two different stock preparations.Proton Inventory Studies on Dark State Retinal Stability and MII Decay/Retinal Release Rates—The stock solutions were prepared as described above and combined in various proportions to give different mole fractions (n) of deuterium ranging from 0-1.0. The dark state thermal decay rate or the MII decay rate of WT rhodopsin was monitored as described above over the range of different mole fractions of D2O. Data were analyzed by plotting the ratio of kn/kHversus the mole fraction of D2O, where kn is the rate in the molar fraction of D2O and kH is the rate in 100% H2O.RESULTSRational for Choice of Mutants—The site-directed rhodopsin mutants analyzed in this study were generated to assess their individual roles in maintaining retinal Schiff base integrity. Specifically, mutants were investigated to evaluate their role in a hydrogen bond network that has previously been proposed based on crystallographic data (23Okada T. Fujiyoshi Y. Silow M. Navarro J. Landau E.M. Shichida Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5982-5987Crossref PubMed Scopus (650) Google Scholar), functional studies (24Yan E.C. Kazmi M.A. Ganim Z. Hou J.M. Pan D. Chang B.S. Sakmar T.P. Mathies R.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9262-9267Crossref PubMed Scopus (182) Google Scholar, 38Janz J.M. Farrens D.L. Vision Res. 2003; 43: 2991-3002Crossref PubMed Scopus (31) Google Scholar, 43Sakmar T.P. Franke R.R. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8309-8313Crossref PubMed Scopus (598) Google Scholar, 44Ramon E. del Valle L.J. Garriga P. J. Biol. Chem. 2003; 278: 6427-6432Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 45Gross A.K. Rao V.R. Oprian D.D. Biochemistry. 2003; 42: 2009-2015Crossref PubMed Scopus (63) Google Scholar, 46Gross A.K. Xie G. Oprian D.D. Biochemistry. 2003; 42: 2002-2008Crossref PubMed Scopus (31) Google Scholar, 47Yan E.C. Kazmi M.A. De S. Chang B.S. Seibert C. Marin E.P. Mathies R.A. Sakmar T.P. Biochemistry. 2002; 41: 3620-3627Crossref PubMed Scopus (86) Google Scholar), and molecular modeling. Each mutant was designed to disrupt the hydrogen bond capability of that individual residue yet preserve the size of the amino acid side chain as much as possible. Toward this end, we made and analyzed the following series of retinal binding pocket mutants: T94I, E113Q, E181Q, S186A, Y192F, and Y268F (Fig. 1). Mutant T94I was investigated not only because of its potential role in this hydrogen bonding network but also because mutant T94I is associated with the disease congenital night blindness (44Ramon E. del Valle L.J. Garriga P. J. Biol. Chem. 2003; 278: 6427-6432Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 45Gross A.K. Rao V.R. Oprian D.D. Biochemistry. 2003; 42: 2009-2015Crossref PubMed Scopus (63) Google Scholar, 46Gross A.K. Xie G. Oprian D.D. Biochemistry. 2003; 42: 2002-2008Crossref PubMed Scopus (31) Google Scholar).Characterization of Rhodopsin Mutants—All mutants expressed to levels similar to WT rhodopsin in a COS cell expression system (10-15 μg/15 cm plate), were capable of binding the chromophore 11-cis-retinal and could be purified following standard procedures to homogeneity. All mutants exhibited spectral ratios (λ280/λmax) between 1.6 and 1.8 with the exception of the counter-ion mutant E113Q, which shows a pH dependence in its absorbance profile (42Sakmar T.P. Franke R.R. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3079-3083Crossref PubMed Scopus (130) Google Scholar, 43Sakmar T.P. Franke R.R. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8309-8313Crossref PubMed Scopus (598) Google Scholar, 48Zhukovsky E.A. Oprian D.D. Science. 1989; 246: 928-930Crossref PubMed Scopus (428) Google Scholar). Many of the mutants exhibited shifted dark state absorption maximum values (λmax), (Fig. 2). Mutant T94I, which is in close proximity to the counter-ion residue Glu113, showed a λmax value of 478 nm in agreement with previous results (44Ramon E. del Valle L.J. Garriga P. J. Biol. Chem. 2003; 278: 6427-6432Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 45Gross A.K. Rao V.R. Oprian D.D. Biochemistry. 2003; 42: 2009-2015Crossref PubMed Scopus (63) Google Scholar, 46Gross A.K. Xie G. Oprian D.D. Biochemistry. 2003; 42: 2002-2008Crossref PubMed Scopus (31) Google Scholar). Interestingly, we also found that mutant T94I could be regenerated with all-trans-retinal, exhibiting a λmax = 464 nm as reported earlier (44Ramon E. del Valle L.J. Garriga P. J. Biol. Chem. 2003; 278: 6427-6432Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Additionally, mutant E181Q showed a slight red shift in its absorbance profile (47Yan E.C. Kazmi M.A. De S. Chang B.S. Seibert C. Marin E.P. Mathies R.A. Sakmar T.P. Biochemistry. 2002; 41: 3620-3627Crossref PubMed Scopus (86) Google Scholar), whereas the dark state λmax of mutant S186A was similar to that of WT rhodopsin (24Yan E.C. Kazmi M.A. Ganim Z. Hou J.M. Pan D. Chang B.S. Sakmar T.P. Mathies R.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9262-9267Crossref PubMed Scopus (182) Google Scholar). Notably, the tyrosine to phenylalanine mutations at residues 192 and 268 resulted in blue shifted λmax values in the dark state of 491 and 495 nm, respectively.Fig. 2UV-visible absorption profiles of purified rhodopsin hydrogen bond network mutants. Photobleaching properties a" @default.
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• W2005661568 title "Role of the Retinal Hydrogen Bond Network in Rhodopsin Schiff Base Stability and Hydrolysis" @default.
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