Short-wavelength visual pigments (SWS1) have lambda(max) values that range from the ultraviolet to the blue. Like all visual pigments, this class has an 11-cis-retinal chromophore attached through a Schiff base linkage to a lysine residue of opsin apoprotein. We have characterized a series of site-specific mutants at a conserved acidic residue in transmembrane helix 3 in the Xenopus short-wavelength sensitive cone opsin (VCOP, lambda(max) approximately 427 nm). We report the identification of D108 as the counterion to the protonated retinylidene Schiff base. This residue regulates the pK(a) of the Schiff base and, neutralizing this charge, converts the violet sensitive pigment into one that absorbs maximally in the ultraviolet region. Changes to this position cause the pigment to exhibit two chromophore absorbance bands, a major band with a lambda(max) of approximately 352-372 nm and a minor, broad shoulder centered around 480 nm. The behavior of these two absorbance bands suggests that these represent unprotonated and protonated Schiff base forms of the pigment. The D108A mutant does not activate bovine rod transducin in the dark but has a significantly prolonged lifetime of the active MetaII state. The data suggest that in short-wavelength sensitive cone visual pigments, the counterion is necessary for the characteristic rapid production and decay of the active MetaII state.

The photobleaching pathway of a short-wavelength cone opsin purified in delipidated form (lambda(max) = 425 nm) is reported. The batho intermediate of the violet cone opsin generated at 45 K has an absorption maximum at 450 nm. The batho intermediate thermally decays to the lumi intermediate (lambda(max) = 435 nm) at 200 K. The lumi intermediate decays to the meta I (lambda(max) = 420 nm) and meta II (lambda(max) = 388 nm) intermediates at 258 and 263 K, respectively. The meta II intermediate decays to free retinal and opsin at >270 K. At 45, 75, and 140 K, the photochemical excitation of the violet cone opsin at 425 nm generates the batho intermediate at high concentrations under moderate illumination. The batho intermediate spectra, generated via decomposing the photostationary state spectra at 45 and 140 K, are identical and have properties typical of batho intermediates of other visual pigments. Extended illumination of the violet cone opsin at 75 K, however, generates a red-shifted photostationary state (relative to both the dark and the batho intermediates) that has as absorption maximum at approximately 470 nm, and thermally reverts to form the normal batho intermediate when warmed to 140 K. We conclude that this red-shifted photostationary state is a metastable state, characterized by a higher-energy protein conformation that allows relaxation of the all-trans chromophore into a more planar conformation. FTIR spectroscopy of violet cone opsin indicates conclusively that the chromophore is protonated. A similar transformation of the rhodopsin binding site generates a model for the VCOP binding site that predicts roughly 75% of the observed blue shift of the violet cone pigment relative to rhodopsin. MNDO-PSDCI calculations indicate that secondary interactions involving the binding site residues are as important as the first-order chromophore protein interactions in mediating the wavelength maximum.

The photochemical and subsequent thermal reactions of the mouse short-wavelength visual pigment (MUV) were studied by using cryogenic UV-visible and FTIR difference spectroscopy. Upon illumination at 75 K, MUV forms a batho intermediate (lambda(max) approximately 380 nm). The batho intermediate thermally decays to the lumi intermediate (lambda(max) approximately 440 nm) via a slightly blue-shifted intermediate not observed in other photobleaching pathways, BL (lambda(max) approximately 375 nm), at temperatures greater than 180 K. The lumi intermediate has a significantly red-shifted absorption maximum at 440 nm, suggesting that the retinylidene Schiff base in this intermediate is protonated. The lumi intermediate decays to an even more red-shifted meta I intermediate (lambda(max) approximately 480 nm) which in turn decays to meta II (lambda(max) approximately 380 nm) at 248 K and above. Differential FTIR analysis of the 1100-1500 cm(-1) region reveals an integral absorptivity that is more than 3 times smaller than observed in rhodopsin and VCOP. These results are consistent with an unprotonated Schiff base chromophore. We conclude that the MUV-visual pigment possesses an unprotonated retinylidene Schiff base in the dark state, and undergoes a protonation event during the photobleaching cascade.

For visual pigments, a covalent bond between the ligand (11-cisretinal) and receptor (opsin) is crucial to spectral tuning and photoactivation. All photoreceptors have retinal bound via a Schiff base (SB) linkage, but only UV-sensitive cone pigments have this moiety unprotonated in the dark. We investigated the dynamics of mouse UV (MUV) photoactivation, focusing on SB protonation and the functional role of a highly conserved acidic residue (E108) in the third transmembrane helix. On illumination, wild-type MUV undergoes a series of conformational changes, batho 3 lumi 3 meta I, finally forming the active intermediate meta II. During the dark reactions, the SB becomes protonated transiently. In contrast, the MUV-E108Q mutant formed significantly less batho that did not decay through a protonated lumi. Rather, a transition to meta I occurred above Ϸ240 K, with a remarkable red shift ( max Ϸ 520 nm) accompanying SB protonation. The MUV-E108Q meta I 3 meta II transition appeared normal but the MUV-E108Q meta II decay to opsin and free retinal was dramatically delayed, resulting in increased transducin activation. These results suggest that there are two proton donors during the activation of UV pigments, the primary counterion E108 necessary for protonation of the SB during lumi formation and a second one necessary for protonation of meta I. Inactivation of meta II in SWS1 cone pigments is regulated by the primary counterion. Computational studies suggest that UV pigments adopt a switch to a more distant counterion, E176, during the lumi to meta I transition. The findings with MUV are in close analogy to rhodopsin and provides further support for the importance of the counterion switch in the photoactivation of both rod and cone visual pigments.V isual pigments are seven transmembrane ␣-helical proteins that initiate the light transduction pathway in retinal photoreceptors. Whereas other G protein-coupled receptors interact with their ligands noncovalently, the visual pigments consist of 11-cis-retinal covalently attached to the apoprotein via a Schiff base (SB) linkage to a conserved lysine in transmembrane helix 7 (TM7) (Fig. 1). After absorption of light, the retinal chromophore isomerizes to the all-trans conformation and triggers a series of conformational changes that lead to the formation of the active state, R* or meta II. All-trans-retinal is eventually released from the vertebrate apoprotein, and the visual pigment can be regenerated with 11-cis-retinal.In dark-adapted rhodopsin, the SB pK a is extraordinarily high resulting in a protonated SB buried within the chromophore binding site (1). The protonation is important to prevent the spontaneous hydrolysis of the SB and contributes to the max (2, 3). The binding site of rhodopsin is neutral (4), and E113 serves as a counterion to the protonated SB (5-8). The stability of the salt bridge is enhanced by a single water molecule that interacts with the side chains of E113 and indirectly through a second water molecule that interacts with adjacent residues (9,...

The role of the extracellular loop region of a short-wavelength sensitive pigment, Xenopus violet cone opsin, is investigated via computational modeling, mutagenesis, and spectroscopy. The computational models predict a complex H-bonding network which stabilizes and connects the EC2-EC3 loop and the N-terminus. Mutations which are predicted to disrupt the H-bonding network are shown to produce visual pigments that do not stably bind chromophore and exhibit properties of misfolded protein. The potential role of a disulfide bond between two conserved Cys residues, Cys105 in TM3 and Cys182 in EC2, is necessary for proper folding and trafficking in VCOP. Lastly, certain residues in the EC2 loop are predicted to stabilize the formation of two anti-parallel β strands joined by a hairpin turn, which interact with the chromophore via H-bonding or Van der Waals interactions. Mutations to conserved residues result in a decrease in chromophore binding. These results demonstrate that the extracellular loops are crucial for the formation of this cone visual pigment. Moreover, there are significant differences in structure and function of this region in VCOP compared to rhodopsin.

The present report deals with an investigation carried out to examine the general validity of characterising the flow pattern inside a swirl chamber atomizer by a modified vortex of the form UR n = a constant over the pressure and discharge range of practical interest with Kerosene as spraying liquid.The principal conclusions that emerge from the investigation are: 1. The exponent 'n' is a function of pressure and atomizer geometry upto a pressure of 400 psi. For a given atomizer geometry, the variation of 'n' with pressure is very nearly linear in this pressure range.2. Beyond 400 psi, the exponent 'n' is a function of only the atomizer geometry. It is independent of pressure in this region.3. The principal geometry parameters which influence the magnitude of 'n' are the orifice diameter, the inlet hole diameter and the swirl chamber diameter.4. It is shown that the value of 'n' may be evaluated with sufficient accuracy from the following correlations:

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