FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2017650111> ?p ?o ?g. }

• W2017650111 endingPage "30166" @default.
• W2017650111 startingPage "30161" @default.
• W2017650111 abstract "Absorption of light in rhodopsin leads through 11-cis- and all-trans-retinal isomerization, proton transfers, and structural changes to the active G-protein binding meta-II state. When meta-II is photolysed by blue light absorption, the activating pathway is apparently reverted, and rhodopsin is photoregenerated. However, the product formed, a P subspecies with A max = 500 nm (P500), is different from the ground state based on the following observations: (i) the ground state fingerprint of 11-cis-retinal does not appear in the infrared spectra, although the proton transfers and structural changes are reverted; (ii) extraction of the retinal from P500 does not yield the expected stoichiometric amount of 11-cis-retinal but predominantly yields all-trans-retinal; (iii) the infrared spectrum of P500 is similar to the classical meta-III intermediate, which arises from meta-II by thermal decay; and (iv) both P500 and meta-III can be photoconverted to meta-II with the same changes in the infrared spectrum and without a significant change in the isomerization state of the extracted chromophore. The data indicate the presence of a “second switch” between active and inactive conformations that operates by photolysis but without isomerization around the C11-C12 double bond. This emphasizes the exclusivity of the ground state, which is only accessible by the metabolic regeneration with 11-cis-retinal. Absorption of light in rhodopsin leads through 11-cis- and all-trans-retinal isomerization, proton transfers, and structural changes to the active G-protein binding meta-II state. When meta-II is photolysed by blue light absorption, the activating pathway is apparently reverted, and rhodopsin is photoregenerated. However, the product formed, a P subspecies with A max = 500 nm (P500), is different from the ground state based on the following observations: (i) the ground state fingerprint of 11-cis-retinal does not appear in the infrared spectra, although the proton transfers and structural changes are reverted; (ii) extraction of the retinal from P500 does not yield the expected stoichiometric amount of 11-cis-retinal but predominantly yields all-trans-retinal; (iii) the infrared spectrum of P500 is similar to the classical meta-III intermediate, which arises from meta-II by thermal decay; and (iv) both P500 and meta-III can be photoconverted to meta-II with the same changes in the infrared spectrum and without a significant change in the isomerization state of the extracted chromophore. The data indicate the presence of a “second switch” between active and inactive conformations that operates by photolysis but without isomerization around the C11-C12 double bond. This emphasizes the exclusivity of the ground state, which is only accessible by the metabolic regeneration with 11-cis-retinal. kiloJoule rhodopsin in the ground state bathorhodopsin Fourier Transform Infrared Spectroscopy lumirhodopsin II, and III, metarhodopsin I, II, and III photoreverted M-II P subspecies with Amax = 470 nm P subspecies with Amax = 500 nm M-II-like species photoreverted from M-III or P heterotrimeric G-protein of the rod cell transducin high pressure liquid chromatography In the ground state of rhodopsin, the chromophore 11-cis-retinal is packed between tight hydrophobic interactions of the β-ionone ring and a salt bridge between the protonated retinal-Schiff base bond to Lys296 and its counterion Glu113 (1Palczewski 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 (5056) Google Scholar). A stable configuration is also adopted with 9-cis- and 7-cis-retinals (2Yoshizawa T. Wald G. Nature. 1963; 197: 1279-1286Crossref PubMed Scopus (380) Google Scholar), suggesting the flexibility of the ground state in adapting to the retinal hydrocarbon chain. After photolysis of the ground state (λmax = 500 nm) by absorption of green light (λ >500 nm), two-thirds of the photonic energy (238 kJ1 mol−1) are taken up in the strained all-trans-isomerized configuration of bathorhodopsin (λmax = 543 nm) (3Cooper A. Nature. 1979; 282: 531-533Crossref PubMed Scopus (181) Google Scholar). The strain is thought to relax through lumirhodopsin (498 nm) by a flip of the retinal β-ionone ring (4Borhan B. Souto M.L. Imai H. Shichida Y. Nakanishi K. Science. 2000; 288: 2209-2212Crossref PubMed Scopus (219) Google Scholar). This movement may trigger the chain of conformational changes leading to metarhodopsin I (M-I = 478 nm) and the signaling state metarhodopsin II (M-II, 380 nm), which binds the G-protein Gt (5Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). The crucial steps in these activating conformational changes are the proton translocation from the retinal-Schiff base to its counterion at Glu113with concurrent breakage of the salt bridge and the subsequent proton uptake that disrupts interactions near Glu134 (5Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 6Sakmar T.P. Prog. Nucleic Acid Res. Mol. Biol. 1998; 59: 1-34Crossref PubMed Scopus (137) Google Scholar). Current evidence suggests that the all-trans-retinal provides a rigid scaffold for the correct adjustment of the structurally sensitive proton translocations (7Meyer C.K. Böhme M. Ockenfels A. Gartner W. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2000; 275: 19713-19718Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Specific determinants of the active state include the β-ionone ring (8Jäger F. Jäger S. Kräutle O. Friedman N. Sheves M. Hofmann K.P. Siebert F. Biochemistry. 1994; 33: 7389-7397Crossref PubMed Scopus (67) Google Scholar), the 9-CH3 group (7Meyer C.K. Böhme M. Ockenfels A. Gartner W. Hofmann K.P. Ernst O.P. J. Biol. Chem. 2000; 275: 19713-19718Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and a non-substituted 10-H group (9Delange F. Boveegeurts P.H.M. Vanoostrum J. Portier M.D. Verdegem P.J.E. Lugtenburg J. DeGrip W.J. Biochemistry. 1998; 37: 1411-1420Crossref PubMed Scopus (61) Google Scholar) of the polyene chain. These three elements are likely to have different interactions in the active versus the ground state (4Borhan B. Souto M.L. Imai H. Shichida Y. Nakanishi K. Science. 2000; 288: 2209-2212Crossref PubMed Scopus (219) Google Scholar, 9Delange F. Boveegeurts P.H.M. Vanoostrum J. Portier M.D. Verdegem P.J.E. Lugtenburg J. DeGrip W.J. Biochemistry. 1998; 37: 1411-1420Crossref PubMed Scopus (61) Google Scholar), suggesting that the chromophore may have different “points of anchor” (10Liu R.S. Mirazadegan T. J. Am. Chem. Soc. 1988; 110: 8617-8623Crossref Scopus (56) Google Scholar, 11Jang G.F. Kuksa V. Filipek S. Bartl F. Ritter E. Gelb M.H. Hofmann K.P.P.K. J. Biol. Chem. 2001; 276: 26148-26153Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) with the protein in the two states. This study arose from the idea to probe the retinal site by light absorption in the active M-II state. When the normal activating pathway is reverted by photolysing M-II with blue light (λ <420 nm), one measures a shift of the absorption maximum indicating reprotonation of the retinal-Schiff base and proton release, a photoproduct with λmax = 500 nm is formed that was so far identified with the 11-cis- or 9-cis-retinal bound ground state (12Matthews R.G. Hubbard R. Brown P.K. Wald G. J. Gen. Physiol. 1963; 47: 215-240Crossref PubMed Scopus (432) Google Scholar, 13Williams T.P. Vision Res. 1968; 8: 1457-1466Crossref PubMed Scopus (26) Google Scholar, 14Arnis S. Hofmann K.P. Biochemistry. 1995; 34: 9333-9340Crossref PubMed Scopus (54) Google Scholar, 15Ernst O.P. Meyer C.K. Marin E.P. Henklein P. Fu W.Y. Sakmar T.P. Hofmann K.P. J. Biol. Chem. 2000; 275: 1937-1943Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 16Grimm C. Reme C.E. Rol P.O. Williams T.P. Invest. Ophthalmol. Visual Sci. 2000; 41: 3984-3990PubMed Google Scholar). However, we will show that this photoproduct does not represent a photoregenerated ground state of rhodopsin or isorhodopsin but rather a product with new properties. Because photoexcitation of the signaling state fails to restore the ground state in vertebrate rhodopsin, the regeneration through the complex cellular metabolism (for review see Ref. 17Palczewski K. Van Hooser J.P. Garwin G.G. Chen J. Liou G.I. Saari J.C. Biochemistry. 1999; 38: 12012-12019Crossref PubMed Scopus (137) Google Scholar) may be the only way to restore 11-cis-retinal bound rhodopsin. Bovine rod outer segments were prepared from fresh dark-adapted retinas by means of a discontinuous sucrose gradient method and stored at −80 °C. Washed rhodopsin membranes were prepared by removing the soluble and membrane-associated proteins from rod outer segment disc membranes by repetitive washes with a low ionic strength buffer and subsequently washed with fatty-acid-free bovine serum albumin (18Sachs K. Maretzki D. Meyer C.K. Hofmann K.P. J. Biol. Chem. 2000; 275: 6189-6194Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 19Pulvermüller A. Schröder K. Fischer T. Hofmann K.P. J. Biol. Chem. 2000; 275: 37679-37685Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The membrane suspension was stored at −80 °C until use. FTIR samples were prepared by centrifugation (20Bartl F. Ritter E. Hofmann K.P. FEBS Lett. 2000; 473: 259-264Crossref PubMed Scopus (32) Google Scholar). In approximately 40 µl of washed membranes (0.3 mm rhodopsin), the pH was adjusted by a few microliters of diluted NaOH or HCl. The suspension was centrifuged for 25 min at 100,000 × g, yielding 2.2 mm rhodopsin in the pellet (from absorption at 500 nm). The buffer solution was removed, and the pellet was transferred to a 30-mm diameter temperature-controlled transmission cell with two BaF2windows and a 5-µm polytetrafluorethylene-gasket. FTIR measurements were performed with a Bruker ifs 66-V spectrometer equipped with an LN2-cooled HgMnTe detector (J15D-series, EG&G Judson). After equilibration for 1 h, a set of 4 transmission spectra was recorded (for details see Ref. 20Bartl F. Ritter E. Hofmann K.P. FEBS Lett. 2000; 473: 259-264Crossref PubMed Scopus (32) Google Scholar). After a 20-s illumination with a 150-watt fiberoptic light source filtered through heat (Schott KG2) and a 495-nm long pass filter, a second set of spectra was recorded. The sets of spectra were averaged, and the M-II minus Rh difference spectrum was generated (we use the convention that the spectra of the conversion A → B are calculated as B − A and termed B − A difference spectrum). The spectra were recorded in the absorbance mode. For photolysis of M-II, the sample was illuminated through a bandpass filter optics (400 ± 20 nm) resulting from the cut-off characteristics of the fiberoptics and a Schott UG1 filter. After illumination for 30 s, the P (photoreverted M-II) minus M-II difference spectrum was generated as described above. To obtain the difference spectra of M-III, an M-II sample was allowed to decay for 2 h at room temperature and at pH 7.0. Then the spectra were taken from the decay product, and the sample was illuminated with green light for 30 s. Subtraction of the decay product spectra (“after” minus “before” illumination) yielded the M-II′ minus M-III difference spectrum. Throughout this study, we use the term M-II′ when an M-II-like species is formed from states other than the rhodopsin ground state. For flash experiments, aliquots of the FTIR preparation were used. M-II was photolysed by a 400-nm light flash (bandwidth is ∼50 nm, Schott BG1) (for details see Ref. 15Ernst O.P. Meyer C.K. Marin E.P. Henklein P. Fu W.Y. Sakmar T.P. Hofmann K.P. J. Biol. Chem. 2000; 275: 1937-1943Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). To follow the formation of P-products, the absorption change at 540 nm was recorded. This wavelength was chosen to minimize the reconversion of the P-products (λmax = 470–500 nm) to M-II′. To monitor the formation of M-II, the samples were photolysed by a 500-nm light flash, and the absorption change was recorded at 380 nm. After recording the infrared spectra, samples (Rh, M-II, or P, see Fig. 1) were removed from the BaF2window of the infrared cuvettes and immediately solved in ice-cold ethanol (21Scherrer P. Mathew M.K. Sperling W. Stoeckenius W. Biochemistry. 1989; 28: 829-834Crossref PubMed Scopus (124) Google Scholar). The solution was stirred for 2 min, and the same amount of heptane was added. After stirring for another 2 min, the sample was centrifuged for 1 min, and the heptane-phase was analyzed immediately in a Hewlett Packard HPLC device (Series 1050) equipped with a Silica Gel 60 column (5 µm) with 5% diethyl ether/heptane at a flow rate of 0.5 ml/min. The traces are of absorption changes at 350 nm and are normalized to total retinal. The procedure to extract retinal oximes was followed as published previously (17Palczewski K. Van Hooser J.P. Garwin G.G. Chen J. Liou G.I. Saari J.C. Biochemistry. 1999; 38: 12012-12019Crossref PubMed Scopus (137) Google Scholar, 22Landers G.M. Olson J.A. J. Chromatogr. 1988; 438: 383-392Crossref PubMed Scopus (49) Google Scholar) but with 10 mm hydroxylamine, 10% SDS, mobile phase, and 12% diethyl ether/heptane. The proportion of each isomer in the samples was determined from the sum of the total peak areas of its syn- andanti-retinal oximes and calculated according to the following extinction coefficients (ε360, in heptane):syn-all-trans, 54,900;anti-all-trans, 51,600;syn-11-cis, 35,000;anti-11-cis, 29,600;syn-13-cis, 49,000; andanti-13-cis, 52,100 (23Groenendijk G.W. de Grip W.J. Daemen F.J. Anal. Biochem. 1979; 99: 304-3410Crossref PubMed Scopus (58) Google Scholar). Concentrated rod disc membrane suspensions were placed in transmission FTIR cuvettes (20Bartl F. Ritter E. Hofmann K.P. FEBS Lett. 2000; 473: 259-264Crossref PubMed Scopus (32) Google Scholar) and either were used directly for UV-visible flash photolysis and FTIR difference spectroscopy or resuspended for retinal extraction and HPLC analysis. To ensure a quantitative extraction with retention of configuration, the retinal extraction was performed in the presence of hydroxylamine (17Palczewski K. Van Hooser J.P. Garwin G.G. Chen J. Liou G.I. Saari J.C. Biochemistry. 1999; 38: 12012-12019Crossref PubMed Scopus (137) Google Scholar). Both the syn and the anti forms of retinaloxime were fully resolved. Fig. 1 A shows HPLC traces of both the native retinaldehydes (left) and of retinaloximes formed with hydroxylamine (right). The data are from the freshly prepared dark-kept samples (Rh), an aliquot after green illumination for 20 s (generating M-II), and an aliquot after green and subsequent blue illumination (generating the photolysis product of M-II termed P). The relative amounts of retinal extracted from these samples were similar for retinal aldehydes and retinal oximes (Fig. 1 A, right). In the fresh membranes, most of the extracted chromophore is in the 11-cis-form with a fraction of approximately 13 ± 3% (maximum deviation in three experiments relative to the total retinal) of all-trans-retinal. The green illumination led to virtually complete conversion into M-II and a correspondingly higher amount (95%) of extracted all-trans-retinal (Fig.1 A). Subsequent blue illumination for 30 s photolysed >60% M-II formed as estimated from the characteristic M-II bands in the FTIR difference spectra (see below). It generated a corresponding amount of “photoreverted” P-product with reprotonated retinal-Schiff base as monitored by the red-shifted absorption maximum in the UV-visible spectra (λmax = 470–500 nm (14Arnis S. Hofmann K.P. Biochemistry. 1995; 34: 9333-9340Crossref PubMed Scopus (54) Google Scholar)) (data not shown). The amount of trans-retinaloxime extracted from the sample after blue light illumination and formation of P-product was slightly lower than in the M-II sample (Fig. 1 A,right). However, the decrease relative to thetrans-isomer formed with M-II was 25% at maximum and not 60% as expected from the amount of the P-product. The fraction ofcis-isomers extracted was 16 and 9% for 11-cis- and 13-cis-retinal, respectively. To minimize the effect of variable amounts of all-trans-retinal in the Rh preparation (freshly prepared dark-kept membranes, Fig. 1 A), the fractions of the retinal isomers were calculated relative to the increase of all-trans-retinal between rhodopsin and M-II. Fig. 1 B shows the FTIR difference spectra of the Rh → M-II and M-II → P conversions measured on the same samples as they were used for HPLC. In the M-II minus Rh difference spectrum (green), a first class of spectral features (protein bands) reflects structural alterations in the protein,i.e. changes in the hydrogen bonding and protonation of carboxyl groups (Asp83, Glu122, and Glu113, at 1768, 1748, and 1712 cm−1, respectively) and in the peptide backbone (amide I and II bands at 1700–1620 and 1570–1500 cm−1, respectively) (24Siebert F. Isr. J. Chem. 1995; 35: 309-323Crossref Scopus (82) Google Scholar). The second class of bands (retinal bands) arises from changes in retinal geometry and retinal-protein interaction reflected in C-C stretching vibrations (fingerprint region, 1238 cm−1 band and its satellites) and hydrogen out of plane vibrations (HOOP region, 960/970 cm−1). Retinal-related bands also appear in the 1550–1570-cm−1 region (C-C stretching vibration), thus interfering with the amide II bands. The difference spectrum of M-II photolysis (blue, normalized to the 1768-cm−1band) expresses the protein bands with inverse polarity, producing a mirror image of the difference spectrum of M-II formation. This does not apply to the retinal C-C stretching vibrations reflecting the geometry of the chromophore (24Siebert F. Isr. J. Chem. 1995; 35: 309-323Crossref Scopus (82) Google Scholar); the fingerprint and HOOP bands are small and show a new pattern that is different from the forward path. Specific for M-II photolysis is a new band at 1350 cm−1, which is only seen with photoreversal and not detected in the forward pathway. This band may reflect new interactions of the reprotonated Schiff base with the receptor environment. Band positions at 1302 cm−1 and 1400 cm−1 were recently assigned to the C-H and N-H in-plane-bending vibrations, respectively, of the retinal chromophore in bacteriorhodopsin (25Heberle J. Fitter J. Sass H.J. Büldt G. Biophys. Chem. 2000; 85: 229-248Crossref PubMed Scopus (69) Google Scholar). Blue flash photolysis of the FTIR samples (Fig.2 A) reproduced the previous finding on suspensions (14Arnis S. Hofmann K.P. Biochemistry. 1995; 34: 9333-9340Crossref PubMed Scopus (54) Google Scholar) that P is a mixture of two products with reprotonated Schiff base, P470 and P500(subscripts indicate λmax). A high affinity C-terminal peptide from the Gt α-subunit inhibits the conversion of M-II to P500, which identifies P470 as a state that does not interact with Gt (15Ernst O.P. Meyer C.K. Marin E.P. Henklein P. Fu W.Y. Sakmar T.P. Hofmann K.P. J. Biol. Chem. 2000; 275: 1937-1943Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). At pH 4.0, the only product formed is P470, which enables the separation of both products in the FTIR spectra. Fig. 2 A shows the spectral differences between M-II and the P-products. The only distinct feature in the P470 minus M-II difference spectrum (Fig.2 A) is the positive band at 1558 cm−1, suggesting that P470 is structurally similar to M-II. This assigns the ensemble of protein bands reflecting deactivation to P500 and fits to the observation that P500 but not P470 is affected by the peptide. Extended blue illumination in photoequilibrium (80% of the initial M-II converted into P-products as defined by the infrared spectrum) produced a steady increase of extracted cis-retinal forms (50% after 20 min, data not shown). At pH 4.0 (where P470 is present), 11-cis-isoform and 13-cis-isoform predominated, whereas at pH 6.0 (with both P470 and P500present), the 9-cis-isoform was seen additionally. During the formation of the cis-species in equilibrium, the infrared spectra of both P500 and P470 did not change significantly. Fig.3 A compares the difference spectra for the conversions M-II → P and P → M-II′ (top,blue and green). The two difference spectra are a perfect mirror image of each other including the fingerprint region ∼1238 cm−1. This shows that chromophore/protein interactions in these two products are reversible in contrast to the rhodopsin → M-II conversion (see Fig. 1 B). The thermal decay of M-II leads in parallel to the hydrolysis of the retinal-Schiff base (yielding opsin and free all-trans-retinal) and to the formation of one or several species with protonated retinal-Schiff base termed metarhodopsin III (M-III, broad UV-visible absorption with λmax ≈ 470 nm). M-III is inactive toward Gt (26Kibelbek J. Mitchell D.C. Beach J.M. Litman B.J. Biochemistry. 1991; 30: 6761-6768Crossref PubMed Scopus (104) Google Scholar). With the formation of M-III, the FTIR protein bands assigned to M-II have disappeared (data not shown) (27Rothschild K.J. Gillespie J. DeGrip W.J. Biophys. J. 1987; 51: 345-350Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 28Klinger A.L. Braiman M.S. Biophys. J. 1992; 63: 1244-1255Abstract Full Text PDF PubMed Scopus (47) Google Scholar). Photolysis of M-III for 30 s with green light reverts up to 40% back to an M-II-like product (M-II′) with a difference spectrum (Fig. 3 A, bottom,green) that shows all essential features of the “reverted reversal” M-II′ minus P spectrum including the protein bands and the fingerprint. Flash illumination (Fig. 3 B) of M-III for 20 µs generates a fast increase of 380 nm absorption. Although this experiment does not identify the product, it is probably a form of M-II, because it is formed with a rate similar to that of M-II formation from the rhodopsin ground state. Also, the efficiency of formation is similar when accounting for the reduced spectral overlap between the excitation spectrum of the flash and M-III as compared with the ground state. Moreover, M-II′ is an active species, which is able to bind G-protein. 2M. Heck, unpublished results.The extraction of retinals from M-III (generated by the decay of M-II for 1.5 h at 20 °C) and M-II′ shows a small shift towardcis-species in the relative weight of retinal isomers that accompanies the formation of M-I′ (Fig. 3 C). The most significant states of the photoreceptor rhodopsin are the light-sensitive ground state and the active state metarhodopsin II, which binds and activates the G-protein. The switch between these two states is operated bycis-/trans-isomerization of the chromophore retinal followed by thermal relaxation and proton transfer reactions. However, it has long been known that the presence of bound all-trans-retinal is compatible with both active and inactive states of the receptor linked to photoproducts M-II and M-III, respectively (26Kibelbek J. Mitchell D.C. Beach J.M. Litman B.J. Biochemistry. 1991; 30: 6761-6768Crossref PubMed Scopus (104) Google Scholar, 29DeGrip W.J. Rothschild K.J. Stavenga D.G. DeGrip W.J. Pugh Jr., E.N. Molecular Mechanism in Visual Transduction. Elsevier Science Publishers B.V., Amsterdam2000: 1-54Google Scholar, 30Hofmann K.P. Stavenga D.G. DeGrip W.J. Pugh Jr., E.N. Molecular Mechanism in Visual Transduction. Elsevier Science Publishers B.V., Amsterdam2000: 99-142Google Scholar). The salient result of this study is that the conversion between M-II- and M-III-like products can also be induced by the absorption of blue or green light, respectively. Although the trigger mechanism of this reversible second switch remains to be elucidated, the results indicate that the underlying chromophore-protein interaction may not involve isomerization around the retinal C11-C12 double bond. The data indicate a reaction scheme as shown in Fig. 4 A. The normal pathway is shown as an outer circle; it comprises the formation of M-II via the Batho and Lumi intermediates, its decay into opsin and all-trans-retinal and the regeneration of ground state rhodopsin from opsin and metabolically supplied 11-cis-retinal. The inner circle contains the thermal decay of M-II into M-III and the photolytic pathways that were newly identified in this study. Here we neglect the “hybrid” product P470, which is M-II-like by its structure but bears a protonated Schiff base. The products P500 and M-III are significantly different in λmax, indicating alterations in chromophore-protein interaction. We have grouped them into the same class of intermediates, because they have in common the photoconversion by green light to an M-II-like product. They are also similar in their infrared spectrum (including the new band at 1350 cm−1), and they both show the largely red-shifted absorption, which indicates a reprotonated retinal-Schiff base. The second switch between the M-III/P500 and M-II/M-II′ products is illustrated in Fig. 4 B. M-III and M-II are separated by a difference in energy of ∼35–40 kJ mol−1, based on ΔH0 of M-I → M-II (42 kJ mol−1(31Cooper A. Converse C.A. Biochemistry. 1976; 15: 2970-2978Crossref PubMed Scopus (60) Google Scholar)) and the ΔG0 between M-I and M-III (∼−8 kJ mol−1 (26Kibelbek J. Mitchell D.C. Beach J.M. Litman B.J. Biochemistry. 1991; 30: 6761-6768Crossref PubMed Scopus (104) Google Scholar)). Thus, the second switch is expected to operate on a shallower energy profile (∼15%) than the ground state and/or Batho activation switch. With the limited time resolution of the available techniques, any conclusions about the mechanism of the deactivation switch rely on a comparison of the starting and final products of thermal decay and photolysis. However, the FTIR spectra have clearly shown that neither the fingerprint nor the hydrogen out of plane bands of the retinal seen in the M-II minus rhodopsin difference spectrum are reverted in the P500 minus M-II or M-II′ minus M-III spectra. This demonstrates that the chromophore-protein interaction in the M-II decay or photolysis products (M-III and P500, respectively) is not the same as in the ground state. It raises the question of which form of the chromophore is actually present in P500, and whether a reversal of the cis- andtrans-isomerization is the trigger that reverses the structural changes seen in the spectra. Not only the spectrometric data but also the retinal extraction data are in conflict with this obvious explanation, because the amount of C11-C12 cis-isomer extracted from P500 was significantly smaller than the stoichiometric. Explanations for this latter finding include: (i) P500contains the C11-C12 cis-isomer, butcis is partially transformed to trans during the extraction procedure; (ii) the conversions between the M-II-like and the M-III-like or P500-like products proceed in a two-photon process involving successive trans- andcis- and cis- andtrans-isomerizations; (iii) the photochemical trigger of the conversion is different from C11-C12 double bond isomerization. Although an extraction artifact (i) cannot be excluded, it is unlikely by the observations made with M-III photolysis. In this case, both the starting and the final product are supposed to bind the retinal in the all-trans-form. And indeed, from both M-III and its photolysis product, M-II′, all-trans-retinal was the predominantly extracted isomer with a slight, if any, shift toward 11-cis- and 7-cis-retinal. In principle, a possible solution could arise from a two-photon process (ii). For example, in the M-III photolyzed in the experiment (Fig. 3), the chromophore could have been transferred from its original binding site to another site in concert with the formation of protonated retinal-Schiff base. Such forms of M-III have indeed been observed (29DeGrip W.J. Rothschild K.J. Stavenga D.G. DeGrip W.J. Pugh Jr., E.N. Molecular Mechanism in Visual Transduction. Elsevier Science Publishers B.V., Amsterdam2000: 1-54Google Scholar). In the two-photon process, a first absorption would isomerize the chromophore. After rapid breakage of the Schiff base bond, the chromophore would return into the original binding site, and a second photon would then photoconvert this newly formed ground state into M-II′. In this study, this possibility was addressed by flash photolysis of M-III. The data are consistent with the notion that M-II′ is formed by flash photolysis from M-III with the same relative efficiency as with continuous illumination. This would limit the time interval between the two photoexcitations in the assumed two-photon process to 20 µs. Although we cannot exclude that low amounts of M-III-like byproducts are formed, most of the M-III species present under our experimental conditions and identified in the FTIR spectra will contain the retinal in its original site or be just “put aside” so that it can readily return into the position it had in M-II. A rough measure for the time it takes to assemble the free retinal chromophore with its binding site is given by the regeneration of rhodopsin from 11-cis-retinal and opsin apoprotein, which takes seconds (18Sachs K. Maretzki D. Meyer C.K. Hofmann K.P. J. Biol. Chem. 2000; 275: 6189-6194Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 32Jäger S. Palczewski K. Hofmann K.P. Biochemistry. 1996; 35: 2901-2908Crossref PubMed Scopus (126) Google Scholar). Therefore, we come to the conclusion that the third mechanism (iii) must be considered a real possibility. The data show that not only 11-cis- but also 7-cis- and 13-cis-isomers are readily detected by the extraction procedure applied. This makes it unlikely that other persistently formed isomers would have escaped the analysis. For the related proton pump bacteriorhodopsin, a light-induced conformational change was found when the protein was regenerated with locked retinal, which cannot isomerize around the C11-C12 double bond. It was discussed that polarization changes in the excited state of the chromophore could trigger a conformational change in the protein that persists even after the chromophore has returned to the ground state (33Aharoni A. Weiner L. Ottolenghi M. Sheves M. J. Biol. Chem. 2000; 275: 21010-21016Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 34Haupts U. Tittor J. Oesterhelt D. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 367-399Crossref PubMed Scopus (507) Google Scholar, 37Rousso I. Khachatryan E. Gat Y. Brodski I. Ottolenghi M. Sheves M. Lewis A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7937-7941Crossref PubMed Scopus (51) Google Scholar). In a recent study on rhodopsin regenerated with locked retinal, it was shown by retinal extraction and HPLC analysis that the chromophore can undergo light-induced isomerization around bonds other than the locked C11-C12 double bond (11Jang G.F. Kuksa V. Filipek S. Bartl F. Ritter E. Gelb M.H. Hofmann K.P.P.K. J. Biol. Chem. 2001; 276: 26148-26153Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In a parallel FTIR analysis, a retinal fingerprint band at 1206 cm−1 appeared that probably reflects the isomerization, but indications of structural changes or changes of the protonation state of carboxyl groups could not be obtained to any significant degree. It was concluded that light-induced changes in the chromophore do occur but cannot induce the activating structural changes in the opsin moiety of the receptor when starting from the protonated Schiff base situation in the ground state (11Jang G.F. Kuksa V. Filipek S. Bartl F. Ritter E. Gelb M.H. Hofmann K.P.P.K. J. Biol. Chem. 2001; 276: 26148-26153Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). A band in the same region appears also in the present reversal or “reverted reversal” spectra (from conversions M-II → P or P/M-III → M-II′, respectively) as part of a chromophore fingerprint, which replaces the normal fingerprint motif at approximately 1238 cm−1 (arising in the forward pathway as a consequence of the steric trigger in Batho and Lumi and persisting through M-I/M-II (24Siebert F. Isr. J. Chem. 1995; 35: 309-323Crossref Scopus (82) Google Scholar)). The presence of the 1206-cm−1 band shows that chromophore alterations other than 11-cis- totrans-isomerization do occur in rhodopsin and that they are reflected in the FTIR spectrum. Because predominantly all-trans-retinal was extracted from the photoreverted species, photoisomerization in the majority of P-product formed can only be transient or metastable. However, the protein is persistently deactivated as seen in the spectral changes and in the blockade of the reversal reactions by bound Gt peptide (Fig. 2). The activating forward pathway of rhodopsin leading to helix motion and the exposure of cytoplasmic interaction sites (5Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 6Sakmar T.P. Prog. Nucleic Acid Res. Mol. Biol. 1998; 59: 1-34Crossref PubMed Scopus (137) Google Scholar, 35Farrens D.L. Altenbach C. Yang K. Hubbell W.L. Khorana H.G. Science. 1996; 274: 768-770Crossref PubMed Scopus (1117) Google Scholar) involves a proton transfer between the protonated Schiff base and its counterion, the side group of Glu-113. This overcomes the structural constraints imposed by the salt bridge between these two locations. The interactions of the retinal β-ionone ring and of the hydrocarbon chain with the protein environment are different in the meta statesversus the ground state configuration (5Okada T. Ernst O.P. Palczewski K. Hofmann K.P. Trends Biochem. Sci. 2001; 26: 318-324Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 8Jäger F. Jäger S. Kräutle O. Friedman N. Sheves M. Hofmann K.P. Siebert F. Biochemistry. 1994; 33: 7389-7397Crossref PubMed Scopus (67) Google Scholar). Anchored in the new interactions and with the Schiff base bond deprotonated, photochemical conversions may be selected that are fundamentally different from those that determine the forward pathway. It will take more extended analyses to find out how this new path proceeds and how similar P500 is to Lumi and/or M-III. On the one hand, the new band at 1350 cm−1 is a distinct property of the photoreversible pathway opened by photolysis of M-II (Figs. 1 and 3) and not seen when starting from the ground state of normal or locked retinal (11Jang G.F. Kuksa V. Filipek S. Bartl F. Ritter E. Gelb M.H. Hofmann K.P.P.K. J. Biol. Chem. 2001; 276: 26148-26153Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). On the other hand, the band at 1206 cm−1, which appeared in the spectra of the rhodopsin with locked retinal (see above), may be a part of the fingerprint motif in the reversal or reverted reversal spectra (Figs. 1 and 3). What we can state is that although a different type of chromophore-protein interaction occurs in M-III or P500 as compared with the ground state, the full set of structural changes appears in the spectra. We propose that in forming or leaving the active state, the different crucial elements are coupled to each other (like the spokes of an umbrella) so that the entity of transformations can be triggered by different mechanisms. Although in vitrotechniques are not sensitive enough to determine the activity of P500 or M-III, it is probable that it is higher than the 11-cis-retinal bound ground state. This would mean that only the 11-cis-isomer can impose the stringent constraints that make the ground state so inactive. The presence of a second rhodopsin-like state is in line with the findings that photoregeneration from early intermediates restores the visual absorption of the ground state but not all of its physiological functions (36Paulsen R. Bentrop J. Nature. 1983; 302: 417-419Crossref PubMed Scopus (27) Google Scholar). Under the conditions of substantial bleaching and dependent on the irradiation conditions, the P-products are expected to accumulate, which may influence bleaching adaptation phenomena. The current observations may also be relevant for the potential physiological role of M-III as a storage form (29DeGrip W.J. Rothschild K.J. Stavenga D.G. DeGrip W.J. Pugh Jr., E.N. Molecular Mechanism in Visual Transduction. Elsevier Science Publishers B.V., Amsterdam2000: 1-54Google Scholar, 30Hofmann K.P. Stavenga D.G. DeGrip W.J. Pugh Jr., E.N. Molecular Mechanism in Visual Transduction. Elsevier Science Publishers B.V., Amsterdam2000: 99-142Google Scholar). The new data underline the exclusivity of the normal photolytic pathway in vertebrate rhodopsin. They show that the ground state, which is relevant for normal green light single quantum detection in the rod cell, cannot be reached by photoreversal. Thus, it must arise from a chemical reaction between 11-cis-retinal and the apoprotein. This may elucidate why it takes a complex metabolic process to regenerate vertebrate rhodopsin. It may be the only possible way to achieve a sufficiently stable chromophore-protein configuration to ensure the exceedingly low noise of the transduction process in rods. We thank Krzysztof Palczewski, Mordechai Sheves, and Oliver P. Ernst for discussions, Peter Henklein for peptide synthesis, and Christine Koch for membrane preparation." @default.
• W2017650111 created "2016-06-24" @default.
• W2017650111 creator A5006554273 @default.
• W2017650111 creator A5021314496 @default.
• W2017650111 creator A5083165090 @default.
• W2017650111 date "2001-08-01" @default.
• W2017650111 modified "2023-10-02" @default.
• W2017650111 title "Signaling States of Rhodopsin" @default.
• W2017650111 cites W1963932033 @default.
• W2017650111 cites W1966589956 @default.
• W2017650111 cites W1974318397 @default.
• W2017650111 cites W1974597502 @default.
• W2017650111 cites W1975297546 @default.
• W2017650111 cites W1975732596 @default.
• W2017650111 cites W1978638198 @default.
• W2017650111 cites W1980823970 @default.
• W2017650111 cites W1982593330 @default.
• W2017650111 cites W1997775954 @default.
• W2017650111 cites W2001526908 @default.
• W2017650111 cites W2004059961 @default.
• W2017650111 cites W2004213037 @default.
• W2017650111 cites W2006255669 @default.
• W2017650111 cites W2013735060 @default.
• W2017650111 cites W2016147733 @default.
• W2017650111 cites W2019742616 @default.
• W2017650111 cites W2027396451 @default.
• W2017650111 cites W2042132016 @default.
• W2017650111 cites W2050192760 @default.
• W2017650111 cites W2056618526 @default.
• W2017650111 cites W2066023824 @default.
• W2017650111 cites W2068370755 @default.
• W2017650111 cites W2081149520 @default.
• W2017650111 cites W2084172527 @default.
• W2017650111 cites W2089090006 @default.
• W2017650111 cites W2103586869 @default.
• W2017650111 cites W2104782686 @default.
• W2017650111 cites W2130049582 @default.
• W2017650111 cites W2149963584 @default.
• W2017650111 cites W2150767772 @default.
• W2017650111 cites W2159019813 @default.
• W2017650111 cites W2159059233 @default.
• W2017650111 doi "https://doi.org/10.1074/jbc.m101506200" @default.
• W2017650111 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11384968" @default.
• W2017650111 hasPublicationYear "2001" @default.
• W2017650111 type Work @default.
• W2017650111 sameAs 2017650111 @default.
• W2017650111 citedByCount "43" @default.
• W2017650111 countsByYear W20176501112012 @default.
• W2017650111 countsByYear W20176501112013 @default.
• W2017650111 countsByYear W20176501112014 @default.
• W2017650111 countsByYear W20176501112015 @default.
• W2017650111 countsByYear W20176501112016 @default.
• W2017650111 countsByYear W20176501112021 @default.
• W2017650111 countsByYear W20176501112022 @default.
• W2017650111 countsByYear W20176501112023 @default.
• W2017650111 crossrefType "journal-article" @default.
• W2017650111 hasAuthorship W2017650111A5006554273 @default.
• W2017650111 hasAuthorship W2017650111A5021314496 @default.
• W2017650111 hasAuthorship W2017650111A5083165090 @default.
• W2017650111 hasBestOaLocation W20176501111 @default.
• W2017650111 hasConcept C185592680 @default.
• W2017650111 hasConcept C202033177 @default.
• W2017650111 hasConcept C2780827179 @default.
• W2017650111 hasConcept C55493867 @default.
• W2017650111 hasConcept C70721500 @default.
• W2017650111 hasConcept C86803240 @default.
• W2017650111 hasConcept C95444343 @default.
• W2017650111 hasConceptScore W2017650111C185592680 @default.
• W2017650111 hasConceptScore W2017650111C202033177 @default.
• W2017650111 hasConceptScore W2017650111C2780827179 @default.
• W2017650111 hasConceptScore W2017650111C55493867 @default.
• W2017650111 hasConceptScore W2017650111C70721500 @default.
• W2017650111 hasConceptScore W2017650111C86803240 @default.
• W2017650111 hasConceptScore W2017650111C95444343 @default.
• W2017650111 hasIssue "32" @default.
• W2017650111 hasLocation W20176501111 @default.
• W2017650111 hasOpenAccess W2017650111 @default.
• W2017650111 hasPrimaryLocation W20176501111 @default.
• W2017650111 hasRelatedWork W1724734852 @default.
• W2017650111 hasRelatedWork W2014852513 @default.
• W2017650111 hasRelatedWork W2069978572 @default.
• W2017650111 hasRelatedWork W2078534481 @default.
• W2017650111 hasRelatedWork W2107192322 @default.
• W2017650111 hasRelatedWork W2115720239 @default.
• W2017650111 hasRelatedWork W2125225930 @default.
• W2017650111 hasRelatedWork W4206917180 @default.
• W2017650111 hasRelatedWork W4206927172 @default.
• W2017650111 hasRelatedWork W4377861143 @default.
• W2017650111 hasVolume "276" @default.
• W2017650111 isParatext "false" @default.
• W2017650111 isRetracted "false" @default.
• W2017650111 magId "2017650111" @default.
• W2017650111 workType "article" @default.