Rhodopsin spectroscopy

Szundi I, Lewis J W and Kliger D S 1997 Deriving reaction mechanisms from kinetic spectroscopy. Application to late rhodopsin intermediates Blophys. J. 73 688-702... 

Brown MF, Salgado GFJ, Struts AV (2010) Retinal dynamics during light activation of rhodopsin revealed by solid-state NMR spectroscopy. BBA-Biomembranes 1798 177-193... 

Nanosecond Absorption Spectroscopy Absorption apparatus, 226, 131 apparatus, 226, 152 detectors, 226, 126 detector systems, 226, 125 excitation source, 226, 121 global analysis, 226, 146, 155 heme proteins, 226, 142 kinetic applications, 226, 134 monochromators/spectrographs, 226, 125 multiphoton effects, 226, 141 nanosecond time-resolved recombination, 226, 141 overview, 226, 119, 147 probe source, 226, 124 quantum yields, 226, 139 rhodopsin, 226, 158 sample holders, 226, 133 singular value decomposition, 226, 146, 155 spectral dynamics, 226, 136 time delay generators, 226, 130. 

Example 2—The first stage in this process of vision has been the excitation of rhodopsin. Rhodopsin partially gets deactivated forming an intermediate, prelumirhodopsin or bathorhodopsin. Picosecond spectroscopy reveals that prelumirhodopsin gets formed because of an intramolecular proton transfer—a jump of a proton from one position to another. 

Spectroscopy and Physical Chemistry of Retinal and Visual Pigments. Several reviews and symposium proceedings discuss the spectroscopic, photochemical, or physicochemical properties of retinal and related compounds, and of natural and model visual pigments derived from them. " " In addition, many papers have been published dealing with specific aspects of the spectroscopy (u.v., n.m.r., resonance Raman) of retinals and rhodopsins" or with aspects of the photochemistry and physical chemistry of retinal derivatives which may be relevant to the functioning of rhodopsin and other visual pigments. The bacterial purple... 

In connection with the problem of oscillations discussed by previous speakers and other types of dynamical behavior of membranes, it would probably be timely to mention here in some more detail the experiments with vision rhodopsin that were performed in our institute by using the Mossbauer spectroscopy method [G. R. Kalamkarov et al., Doklady Biophys., 219, 126 (1974)]. These experiments manifested the existence of reversible photo-induced conformational changes in the photoreceptor membrane even at such low temperature as 77°K. We have labeled various samples of solubilized rhodopsin and of photoreceptor membranes by iron ascorbate enriched with Fe57 and looked for the change of Mossbauer spectra caused by the illumination of our samples. 

The Photoactive Yellow Protein (PYP) is the blue-light photoreceptor that presumably mediates negative phototaxis of the purple bacterium Halorhodospira halophila [1]. Its chromophore is the deprotonated trans-p-coumaric acid covalently linked, via a thioester bond, to the unique cystein residue of the protein. Like for rhodopsins, the trans to cis isomerization of the chromophore was shown to be the first overall step of the PYP photocycle, but the reaction path that leads to the formation of the cis isomer is not clear yet (for review see [2]). From time-resolved spectroscopy measurements on native PYP in solution, it came out that the excited-state deactivation involves a series of fast events on the subpicosecond and picosecond timescales correlated to the chromophore reconfiguration [3-7]. On the other hand, chromophore H-bonding to the nearest amino acids was shown to play a key role in the trans excited state decay kinetics [3,8]. In an attempt to evaluate further the role of the mesoscopic environment in the photophysics of PYP, we made a comparative study of the native and denatured PYP. The excited-state relaxation path and kinetics were monitored by subpicosecond time-resolved absorption and gain spectroscopy. 

To summarize, Jean shows that coherence can be created in a product as a result of nonadiabatic curve crossing even when none exists in the reactant [24, 25]. In addition, vibrational coherence can be preserved in the product state to a significant extent during energy relaxation within that state. In barrierless processes (e.g., an isomerization reaction) irreversible population transfer from one well to another occurs, and coherent motion can be observed in the product regardless of whether the initially excited state was prepared vibrationally coherent or not [24]. It seems likely that these ideas are crucial in interpreting the ultrafast spectroscopy of rhodopsins [17], where coherent motion in the product is directly observed. Of course there may be many systems in which relaxation and dephasing are much faster in the product than the reactant. In these cases lack of observation of product coherence does not rule out formation of the product in an essentially ballistic manner. 

In a crystal structure47111-ds-retinal has the 12-s-cis conformation shown at the top in Eq. 23-36 rather than the 12-s-frans conformation at the center and in which there is severe steric hindrance between the 10-H and 13-CH3. Nevertheless, H-and 13C-NMR spectroscopy suggest that the retinal in rhodopsin is in a twisted 12-s-frans conformation.472 4723 The Schiff base of 11-ds-retinal with N-butylamine has an absorption maximum at -360 nm but N-protonation, as in the structure in Eq. 23-36, shifts the maximum to 440 nm with emax = 40,600 M 1 cm 1 (Fig. 23-42). This large shift in the wavelength of the absorption maximum (the opsin shift) indicates that binding to opsin stabiliz-... 

The reaction sequence of Eq. 23-37 can be slowed by lowering the temperature. Thus, at 70K illumination of rhodopsin leads to a photostationary state in which only rhodopsin, bathorhodopsin, and a third form, isorhodopsin, are present in a constant ratio.510 Isorhodopsin (maximum absorption at 483 nm)513 contains 9-ds-retinal and is not on the pathway of Eq. 23-37. Resonance Raman spectroscopy at low temperature supports a distorted all-frans structure for the retinal Schiff base in bathorhodopsin.510 The same technique suggests the trans geometry of the C = N bond shown in Eqs. 23-38 and 23-39. Simple Schiff bases of 11-cz s-retinal undergo isomerization just as rapidly as does rhodopsin.514... 

Dunham, T. D., and Farrens, D. L. (1999). Conformational changes in rhodopsin. Movement of helix f detected by site-specific chemical labeling and fluorescence spectroscopy./. Biol. Chem. 274, 1683-1690. 

A great deal of fundamental information about vision has been obtained through absorption spectroscopy. 6) The primary event in vision is the photo-chemical formation of bathos rhodopsin from rhodopsin and isorhodopsin. Rhodopsin is the... 

Retinal as Visual Pigment Model Spectroscopy and Physical Chemistry. Several theoretical, spectroscopic, and photochemical studies aimed at correlating the behaviour of retinal and derivatives, especially Schiff s bases, with that of rhodopsin and related visual pigments have been reported.95-104 A review of recent work in this field has been presented.105... 

In spite of the differences in the behavior of the pigments, in situ, as compared to extracts, studies on extracts provide useful information, not only on the structure of the bleaching intermediates, but also on the possible roles they may be playing in the photoreceptors. The bleaching intermediates from several species have been investigated extensively by using flash photolysis techniques and both low temperature and ultrafast kinetic spectroscopy. As an example, Fig. 5 shows the sequence of the intermediates in the photolysis of bovine and squid rhodopsin extracts. 

Resonance Raman and NMR Studies. The major support to the protonation hypothesis is presently based on the recent application of resonance-Raman spectroscopy. (For recent reviews, see refs. 217-219.) The method uses an incident beam which is in resonance with the absorption of the retinyl chromophore. This results in the selective enhancement of the Raman cross sections coupled with the chromophore, relative to the very weak, non-resonant, modes of the opsin. Characteristic spectra are shown in Fig. 6. Early evidence for protonation came from the observation of a close similarity between the C=N vibrational frequency in rhodopsin and in a model protonated Schiff base (220). More conclusive arguments were provided by Oseroff and Callender, who carried out experiments at low temperatures in order to control sample photoability (221). It was observed that deuteration shifts the C=N vibration frequency from 1655 cm- to 1630 cm-- -, both in the pigment and in a model protonated Schiff base. 

Figure 8. Ultrafast processes associated with bathorhodopsin and hypsorhodopsin monitored by picosecond absorption spectroscopy. (A) Absorbance changes (aA) as function of time monitored at 530 nm, showing the decay of a species (PBAT) identified as the precursor of BAT. (B) Arrhenius plot for the rate constant of this process in rhodopsin and in deuterated rhodopsin. (C) Decay of hypsorhodopsin. (D) Formation of bathorhodopsin. [(A) and (B) data from ref. 301 for bovine rhodopsin (in 0.1 M Ammonix LO, 66% ethylene glycol, at pH 7.0). (C) and (D) data from ref. 298 for squid rhodopsin in 2% digitonin (pH 10.5) at room temperature.]...

Photoreceptor Pigments. There have been several reviews on the structures, photochemistry, and functioning of the retinal-protein photoreceptor pigments involved in the processes of visionand in the purple membrane of Halobacteria (bacteriorhodopsin). ° ° In addition to the papers quoted earlier on the spectroscopy of these pigments, many other reports have appeareddealing with rhodopsin and intermediates in its photocycle, especially photochemistry, chromophore-protein conformation and binding, and reaction kinetics. Similar studies on bacteriorhodopsin have also been described." "-"" ... 

Werner K, et al. Isotope labeling of mammalian GPCRs in 67. HEK293 cells and characterization of the C-terminus of bovine rhodopsin by high resolution liquid NMR spectroscopy. J. 68. Biomolec. NMR 2008 40 49-53. 

Upon illumination bacteriorhodopsin undergoes a series of interconversions which are detectable by following changes in the position and amplitude of its absorption band. The photointermediates are designated by a letter (in analogy with photoproducts earlier described for visual rhodopsin), numbered with the wavelength of maximal absorption [34]. Time-resolved spectroscopy after flashes, and low tempera-... 

Studies of TM residue accessibility to water-soluble sulfhydryl-reactive compounds has allowed Javitch and coworkers to gain evidence that P2-AR activation also involves a conformational rearrangement of TM VI (123), whereas detailed fluorescence spectroscopy studies by Kobilka and coworkers indicated that the helical movements occurring with P2-AR activation are almost identical to those for rhodopsin, that is, a counterclockwise rotation (when viewed from the extracellular surface of the receptor) of both TM III and TM VI, with a tilting of the cytoplasmic end of the latter toward TM V (74,124). The importance of the orientation of TM VI, which is stabilized by interhelical interactions with TM V, comes from studies of the arARs, which showed that mutation of either the... 

Jager S, Lewis JW, Zvyaga TA, Szundi I, Sakmar TP, Kliger DS. Time-resolved spectroscopy of the early photolysis intermediates of rhodopsin Schiff base counterion mutants. Biochemistry 1997 36 1999-2009. 

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