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The higher quantum yield of mouse UV than the unprotonated form of the rhodopsin E113Q mutant suggests that mouse UV has specific mechanism for efficient photoisomerization of the unprotonated Schiff base. Schiff base protonation plays a role in increasing quantum yield.

An investigation is done to find out how the chloride binding affects the structure and electronic state of the chromophore of iodopsin and bathoiodopsin. The results shows that iodopsin's chromophere has a unique structure near its center and that the binding of chloride nearby enhances the difference.

A cooled CCD spectrophotometer with a time resolution of 9.7 ms is developed to identify meta-Ib in rod outer segment (ROS) membranes at physiological temperature. The results show that meta-Ib exists under physiological conditions and it is found that meta-Ib interacts with G-protein transducin (Gt) even in the presence of excess GDP such that extra-meta-II is no longer formed.

Research examines the molecular mechanism underlying the molecular properties between the rhodopsin and cone pigments. Results indicate that differences in the meta II decay rate between the chicken cone pigments and rhodopsin is due to differences in the amino acid residues at 189 and 122.

Research has been conducted on the rod and cone visual pigments. The mechanisms and specific amino acids responsible for the differences in molecular properties of these pigments have been investigated.

Results demonstrate that chloride binding induces conformational changes in iodopsin and the binding site interacts with the protein's chromophore upon photoisomerization. Data indicate that water molecules at the site form a hydrogen-bonding network with amino acid residues causing a red shift in the absorption spectrum of the protein.

The chimeric nature of pinopsin between rod and cone visual pigments is discussed. Complete photoreversibility was seen in pinopsin, bathopinopsin and isopinopsin. Light absorbed by pinopsin brings on the first step of cis-trans isomerization of the 11-cis-retinylidene chromophore at -196 degrees C. The meta II formation kinetics of pinopsin is comparable to those of a cone pigment The decay profile is much like that of rhodopsin. The unique photoreaction is the first example of the chimeric nature of rod- and cone-type pigments in vertebrates.

Research was conducted to examine the impact of anion binding on iodopsin, the chicken red-sensitive cone visual pigment, via calculations of the Fourier transform infrared spectra of chloride- and nitrate-bound forms of iodopsin at 77 K. From the results, the molecular mechanism of the spectral red shift and that leading to the unique thermal behavior of bathoiodopsin were analyzed.

It appears that the intermediate formed before meta II during the photobleaching process of rhodopsin also binds to transducin. However, it does not induce the GDP-GTP exchange reaction in transducin. A new intermediate known as meta Ib exists between metarhodopsin I (meta Ia) and metarhodopsin II (meta II). This intermediate binds only to transducin, while meta II catalyzes a GDP-GTP exchange in transducin.

Comparison of the molecular properties of the visual pigments chicken blue, chicken green, chicken red and rhodopsin found that the faster decay of enzymatically active state is common in cone visual pigments and is independent of their spectral sensitivity. Data indicate that retinal chromophore changes its position relative to the amino acid residues during the lumi-to-meta I transition. These residues are those that regulate the maxima of original pigments through electrostatic interactions. Other residues regulate the thermal reactions of the visual pigments.

A comparison of the thermal reactions of the batho and meta I intermediates in the bleaching of iodopsin, a red-sensitive cone visual pigment, and of the chloride-bound and unbound iodopsins indicates that the binding of chloride is necessary for the reverse reactions of the meta I intermediate. The reverse reaction from the batho do not require chloride binding. A study of the reverse reaction of meta I with respect to chloride concentration reveals that the dissociation constant of meta I is 200 times higher than iodopsin.

The intermediates formed by the photobleaching of the chicken green, a green-sensitive cone visual pigment, are less stable than those of rhodopsin because of smaller activation enthalpies. The meta intermediates are more stable in alkaline conditions. The wavelengths of the absorption maxima of chicken green meta intermediaries are shorter than those of rhodopsin. However the amino acid sequence and the absorption spectrum of these visual pigments is similar. The difference in the properties of chicken green are controlled by dissociative amino acid residues.

Low temperature spectroscopy enables an analysis of the photobleaching processes in gecko blue and gecko green, two visual pigments form the retina of Tokay gecko, which are purified by column chromatography. The photobleaching phenomenon in gecko green constitutes six intermediates, of which three are meta I, meta II and meta III. These three intermediates are present in the photobleaching process of gecko blue also. The meta II intermediate of both the pigments is thermally less stable than metarhodopsin II. The results indicate a cone-type structure for both the gecko visual pigments.

Low-temperature time-resolved spectroscopic analysis confirm the existence of thermal equilibria among metarhodopsin I(meta I), lumirhodopsin (lumi) and metarhodopsin II (meta II) in chicken rhodopsin. High energy absorbed by the chromophore is converted via the meta I stage to induce conformational changes in the protein, ultimately forming meta II. Lumi and meta II have larger enthalpy and entropy than meta I, but have nearly similar thermodynamic parameters.

Studies of the molecular properties of chicken green and comparison with rhodopsin reveal that chicken green molecular extinction coefficient and photosensitivity are the same as that of rhodopsin. Chicken green is faster than rhodopsin in terms of regeneration from opsin moieties and the chromophore 11-cis-retinal and formation of the physiologically-active intermediate. These differences are similar to the physiological differences that exist between cones and rods.

The quantum yield of E113Q mutant of the E113 in the UV-absorbing visual pigments of vertebrates is found to be lower than the normal animals. This demonstrates the role of E113 residue in the photoisomerization of the unprotonated chromophores for effective absorption of light by the retinal cells.

An investigation is conducted on the identification of amino acid residues to the regulation of the meta-III intermediate decay rate. It is shown that the meta-III intermediates exhibit about 700 times difference in decay times between the two pigments, the chicken rhodopsin and green-sensitive cone pigments, and the faster decay in chicken green can be converted to the slower decay in rhodopsin by replacing the residues in chicken green with the corresponding rhodopsin residues.

The thermal reactions of the bathoproduct of the long wavelength sensitive visual pigment iodopsin are investigated. It was found that iodopsin exhibits two conformational states, each of which is stabilized by the binding of chloride and nitrate, respectively.

Protonation of the retinal Schiff base chromophore is responsible for the absorption of visible light and is stabilized by the counterion residue E113 in vertebrate visual pigments. However, this residue is also conserved in vertebrate UV-absorbing visual pigments (UV pigments) which have an unprotonated Schiff base chromophore. To elucidate the role played by this residue in the photoisomerization of the unprotonated chromophore in UV pigments, we measured the quantum yield of the E113Q mutant of the mouse UV cone pigment (mouse UV). The quantum yield of the mutant was much lower than that of the wild type, indicating that E113 is required for the efficient photoisomerization of the unprotonated chromophore in mouse UV. Introduction of the E113Q mutation into the chicken violet cone pigment (chicken violet), which has a protonated chromophore, caused deprotonation of the chromophore and a reduction in the quantum yield. On the other hand, the S90C mutation in chicken violet, which deprotonated the chromophore with E113 remaining intact, did not significantly affect the quantum yield. These results suggest that E113 facilitates photoisomerization in both UV-absorbing and visible light-absorbing visual pigments and provide a possible explanation for the complete conservation of E113 among vertebrate UV pigments. [ABSTRACT FROM AUTHOR]

Studies the presence of thermal equilibrium among variants of rhodopsin. Back reactions from metarhodopsin to lumirhodopsin; Sample preparation; Measurement of spectral changes; Estimation of relative thermodynamic parameters.

Ligand-triggered activation of G protein-coupled receptors (GPCRs) relies on the phenomenon of loose allosteric coupling, which involves conformational alterations spanning from the extracellular ligand-binding domain to the cytoplasmic region, where interactions with G proteins occur. During the GPCR activation process, several intermediate and equilibrium states orchestrate the movement of the flexible and rigid transmembrane (TM) segments of the GPCR. Monitoring early conformational changes is important in unraveling the structural intricacies of the loose allosteric coupling. Here, we focus on the lumi intermediate formed by thermal relaxation from the initial photointermediate, batho in primate green cone pigment (MG), a light-sensitive GPCR responsible for color vision. Our findings from light-induced Fourier transform infrared difference spectroscopy reveal its similarity with rhodopsin, which mediates twilight vision, specifically involving the flip motion of the β-ionone ring, the relaxation of the torsional structure of the retinal, and local perturbations in the α-helix upon lumi intermediate formation. Conversely, we observe a hydrogen bond modification specific to MG's protonated carboxylic acid, identifying its origin as Glu102 2.53 situated in TM2. The weakening of the hydrogen bond strength at Glu102 2.53 during the transition from the batho to the lumi intermediates corresponds to a slight outward movement of TM2. Additionally, within the X-ray crystal structure of the rhodopsin lumi intermediate, we note the relocation of the Met86 2.53 side chain in TM2, expanding the volume of the retinal binding pocket. Consequently, the position of 2.53 emerges as the early step in the conformational shift toward light-induced activation. Moreover, given the prevalence of IR-insensitive hydrophobic amino acids at position 2.53 in many rhodopsin-like GPCRs, including rhodopsin, the hydrogen bond alteration in the C═O stretching band at Glu102 2.53 of MG can be used as a probe for tracing conformational changes during the GPCR activation process.

To investigate the chromophore structures of iodopsin and its low-temperature photoproducts, we have assigned their vibrational bands in the Fourier transform infrared (FTIR) spectra using iodopsin samples that were reconstituted with a series of (13)C- and deuterium-labeled retinals. The analyses of the vibrational bands in the fingerprint and hydrogen-out-of-plane (HOOP) regions indicated that the structure of the chromophores in the iodopsin system differs near their centers from those in the rhodopsin system. Compared to rhodopsin, the chromophore of the batho intermediate of iodopsin is twisted in the C(12) to C(14) regions but is more planar around C(11) region. The large amount of twisting was reduced by removing the chloride ion from the iodopsin, suggesting that this twisting hinders the relaxation of the torsion near C(11) necessary for the transition to the lumi intermediate and thus results in the thermal reversion of the batho intermediate back to the iodopsin. From the analyses of the C=NH and C=ND stretching bands, we conclude that the displacement of the Schiff base region upon photoisomerization of the chromophore is restricted, as is the case for rhodopsin. These results indicated that iodopsin's chromophore has a unique structure near its center and that this difference is enhanced by the binding of chloride nearby.