Wavelength dependent cis-trans isomerization in vision.

The primary event in vision is the light-driven cis-trans isomerization of the 11-cis-retinal chromophore in the G-protein coupled receptor rhodopsin. Early measurements showed that this photoisomerization has a reaction quantum yield phi of approximately 0.67 [Dartnall (1936) Proc. R. Soc. A 156, 158-170; Dartnall (1968) Vision Res. 8, 339-358] and suggested that the quantum yield was wavelength independent [Schneider (1939) Proc. Natl. Acad. Sci. U.S.A. 170, 102-112]. Here we more accurately determine phi(500) = 0.65 +/- 0.01 and reveal that phi surprisingly depends on the wavelength of the incident light. Although there is no difference in the quantum yield between 450 and 480 nm, the quantum yield falls significantly as the photon energy is reduced below 20 000 cm(-1) (500 nm). At the reddest wavelength measured (570 nm), the quantum yield is reduced by 5 +/- 1% relative to the 500 nm value. These experiments correct the long-held presumption that the quantum yield in vision is wavelength independent, and support the hypothesis that the 200 fs photoisomerization reaction that initiates vision is dictated by nonstationary excited-state vibrational wave packet dynamics.     

Euphausiid visual pigments. The rhodopsins of Euphausia superba and Meganyctiphanes norvegica (Crustacea, Euphausiacea)

The rhabdoms of Euphausia superba contain one digitonin-extractable rhodopsin, lambda max 485 nm. The rhodopsin undergoes unusual pH- dependent spectral changes: above neutrality, the absorbance decreases progressively at 485 nm and rises near 370 nm. This change is reversible and appears to reflect an equilibrium between a protonated and an unprotonated form of the rhodopsin Schiff-base linkage. Near neutral pH and at 10 degrees C, the rhodopsin is partiaLly converted by 420-nm light to a stable 493-nm metarhodopsin. The metarhodopsin is partially photoconverted to rhodopsin by long-wavelength light in the absence of NH2OH; in the presence of NH2OH, it is slowly converted to retinal oxime and opsin. The rhodopsin of Meganyctiphanes norvegica measured in fresh rhabdoms by microspectrophotometry has properties very similar to those of the extracted rhodopsin of E. superba. Its lambda max is 488 nm and it is partially photoconverted by short wavelength irradiation to a stable photoconvertible metarhodopsin similar to that of E. superba. In the presence of light and NH2OH, the M. norvegica metarhodopsin is converted to retinal oxime and opsin. Our results indicate that previous determinations of euphausiid rhodopsin absorbance spectra were incorrect because of accessory pigment contamination.     

Femtosecond formation dynamics of primary photoproducts of visual pigment rhodopsin

The coherent 11-cis-retinal photoisomerization dynamics in bovine rhodopsin was studied by femtosecond time-resolved laser absorption spectroscopy at 30-fs resolution. Femtosecond pulses of 500, 535, and 560 nm wavelength were used for rhodopsin excitation to produce different initial Franck-Condon states and relevant distinct values of the vibrational energy of the molecule in its electron excited state. Time evolution of the photoinduced rhodopsin absorption spectra was monitored after femtosecond excitation in the spectral range of 400–720 nm. Oscillations of the time-resolved absorption signals of rhodopsin photoproducts represented by photorhodopsin₅₇₀ with vibrationally-excited all-trans-retinal and rhodopsin₄₉₈ in its initial state with vibrationally-excited 11-cis-retinal were studied. These oscillations reflect the dynamics of coherent vibrational wave-packets in the ground state of photoproducts. Fourier analysis of these oscillatory components has revealed frequencies, amplitudes, and initial phases of different vibrational modes, along which the motion of wave-packets of both photoproducts occurs. The main vibrational modes established are 62, 160 cm⁻¹ and 44, 142 cm⁻¹ for photorhodopsin₅₇₀ and for rhodopsin₄₉₈, respectively. These vibrational modes are directly involved in the coherent reaction under the study, and their amplitudes in the power spectrum obtained through the Fourier transform of the kinetic curves depend on the excitation wavelength of rhodopsin.     

The nature of the primary photochemical events in rhodopsin and isorhodopsin.

The nature of the primary photochemical events in rhodopsin and isorhodopsin is studied by using low temperature actinometry, low temperature absorption spectroscopy, and intermediate neglect of differential overlap including partial single and double configuration interaction (INDO-PSDCI) molecular orbital theory. The principal goal is a better understanding of how the protein binding site influences the energetic, photochemical, and spectroscopic properties of the bound chromophore. Absolute quantum yields for the isorhodopsin (I) to bathorhodopsin (B) phototransformation are assigned at 77 K by using the rhodopsin (R) to bathorhodopsin phototransformation as an internal standard (phi R----B = 0.67). In contrast to rhodopsin photochemistry, isorhodopsin displays a wavelength dependent quantum yield for photochemical generation of bathorhodopsin at 77 K. Measurements at seven wavelengths yielded values ranging from a low of 0.089 +/- 0.021 at 565 nm to a high of 0.168 +/- 0.012 at 440 nm. An analysis of these data based on a variety of kinetic models suggests that the I----B phototransformation encounters a small activation barrier (approximately 0.2 kcal mol-1) associated with the 9-cis----9-trans excited-state torsional-potential surface. The 9-cis retinal chromophore in solution (EPA, 77 K) has the smallest oscillator strength relative to the other isomers: 1.17 (all-trans), 0.98 (9-cis), 1.04 (11-cis), and 1.06 (13-cis). The effect of conformation is quite different for the opsin-bound chromophores. The oscillator strength of the lambda max absorption band of I is observed to be anomalously large (1.11) relative to the lambda max absorption bands of R (0.98) and B (1.07). The wavelength-dependent photoisomerization quantum yields and the anomalous oscillator strength associated with isorhodopsin provide important information on the nature of the opsin binding site. Various models of the binding site were tested by using INDO-PSDCI molecular orbital theory to predict the oscillator strengths of R, B, and I and to calculate the barriers and energy storage associated with the photochemistry of R and I for each model. Our experimental and theoretical investigation leads to the following conclusions: (a) The counterion (abbreviated as CTN) is not intimately associated with the imine proton in R, B, or I. The counterion lies underneath the plane of the chromophore in R and I, and the primary chromophore-counterion electrostatic interactions involve C15-CTN and C13-CTN. These interactions are responsible for the anomalous oscillator strength of I relative to R and B.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coexpression of spectrally distinct rhodopsins in Aedes aegypti R7 photoreceptors

The retina of the mosquito Aedes aegypti can be divided into four regions based on the non-overlapping expression of a UV sensitive Aaop8 rhodopsin and a long wavelength sensitive Aaop2 type rhodopsin in the R7 photoreceptors. We show here that another rhodopsin, Aaop9, is expressed in all R7 photoreceptors and a subset of R8 photoreceptors. In the dorsal region, Aaop9 is expressed in both the cell body and rhabdomere of R7 and R8 cells. In other retinal regions Aaop9 is expressed only in R7 cells, being localized to the R7 rhabdomere in the central and ventral regions and in both the cell body and rhabdomere within the ventral stripe. Within the dorsal-central transition area ommatidia do not show a strict pairing of R7-R8 cell types. Thus, Aaop9 is coexpressed in the two classes of R7 photoreceptors previously distinguished by the non-overlapping expression of Aaop8 and Aaop2 rhodopsins. Electroretinogram analysis of transgenic Drosophila shows that Aaop9 is a short wavelength rhodopsin with an optimal response to 400-450 nm light. The coexpressed Aaop2 rhodopsin has dual wavelength sensitivity of 500-550 nm and near 350 nm in the UV region. As predicted by the spectral properties of each rhodopsin, Drosophila photoreceptors expressing both Aaop9 and Aaop2 rhodopsins exhibit a uniform sensitivity across the broad 350-550 nm light range. We propose that rhodopsin coexpression is an adaptation within the R7 cells to improve visual function in the low-light environments in which Ae. aegypti is active.     

The photobleaching sequence of a short-wavelength visual pigment.

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.     

Squid retinochrome

Retinochrome is a photosensitive pigment located primarily in the inner portions of the visual cells of cephalopods. Its absorption spectrum resembles that of rhodopsin, but its chromophore is all-trans retinal, which light isomerizes to 11-cis, the reverse of the situation in rhodopsin. The 11-cis photoproduct of retinochrome slowly reverts to retinochrome in the dark. The chromophoric site of retinochrome is more reactive than that of most visual pigments: (a) Hydroxylamine converts retinochrome in the dark to all-trans retinal oxime + retinochrome opsin. (by Sodium borohydride reduces it to N-retinyl opsin. (c) Lambda max of retinochrome shifts from 500 to 515 nm as the pH is raised from 6 to 10, with a loss of absorption above pH 8; meanwhile above this PH a second band appears at shorter wavelengths with lambda max 375 nm. These changes are reversible. (d) If retinochrome is incubated with all-trans 3-dehydroretinal (retinal2) in the dark, some 3-dehydroretinochrome (retinochrome2, lambda max about 515 nm) is formed. Conversely, when retinochrome2, made by adding all-trans retinal2 to bleached retinochrome or retinochrome opsin, is incubated in the dark with all-trans retinal some of it is converted to retinochrome. Retinal and 3-dehydroretinal therefore can replace each other as chromophores in the dark.     

Regeneration of bovine and octopus opsins in situ with natural and artificial retinals

We consider the problem of color regulation in visual pigments for both bovine rhodopsin (lambda max = 500 nm) and octopus rhodopsin (lambda max = 475 nm). Both pigments have 11-cis-retinal (lambda max = 379 nm, in ethanol) as their chromophore. These rhodopsins were bleached in their native membranes, and the opsins were regenerated with natural and artificial chromophores. Both bovine and octopus opsins were regenerated with the 9-cis- and 11-cis-retinal isomers, but the octopus opsin was additionally regenerated with the 13-cis and all-trans isomers. Titration of the octopus opsin with 11-cis-retinal gave an extinction coefficient for octopus rhodopsin of 27,000 +/- 3000 M-1 cm-1 at 475 nm. The absorption maxima of bovine artificial pigments formed by regenerating opsin with the 11-cis dihydro series of chromophores support a color regulation model for bovine rhodopsin in which the chromophore-binding site of the protein has two negative charges: one directly hydrogen bonded to the Schiff base nitrogen and another near carbon-13. Formation of octopus artificial pigments with both all-trans and 11-cis dihydro chromophores leads to a similar model for octopus rhodopsin and metarhodopsin: there are two negative charges in the chromophore-binding site, one directly hydrogen bonded to the Schiff base nitrogen and a second near carbon-13. The interaction of this second charge with the chromophore in octopus rhodopsin is weaker than in bovine, while in metarhodopsin it is as strong as in bovine.     

Excitation signal processing times in Halobacterium halobium phototaxis.

Phototaxis responses of Halobacterium halobium were monitored with a computerized cell-tracking system coupled to an electronic shutter controlling delivery of photostimuli. Automated analysis of rates of change in direction and linear speeds provided detection of swimming reversals with 67 ms resolution, permitting measurement of distinct phases of the responses to attractant and repellent stimuli. After stimulation, there was a latency period in which the population reversal frequency was unchanged, followed by an excitation phase in which reversal frequency increased, and a slower adaptation phase in which reversal frequency returned to its prestimulus value. A step-decrease in illumination of the attractant receptor slow-cycling or sensory rhodopsin (SR) (lambda max, 587 nm) was interpreted by the cells as an unfavorable stimulus and, after a minimum latency of 0.70 +/- 0.14 s, induced swimming reversals with the peak response occurring 1.34 +/- 0.07 s after onset of the stimulus. Two distinct repellent responses in the near UV/blue were observed. One was a reversal response to 400 nm light, which was dependent on orange-red background illumination as expected for the photointermediate repellent form of SR (lambda max, 373 nm). The minimum latency of this response was approximately the same as that of the SR attractant system. The second was a reversal response with shorter minimum latency (0.40 +/- 0.07 s) to light of longer wavelength (450 nm) than absorbed by the known SR repellent form. This result confirms recent findings of an additional repellent photosystem in this spectral range. Further, the longer wavelength repellent response is independent of orange-red background illumination, indicating that the photoreceptor mediating this response is not a photointermediate of SR.     

Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence

The mechanism of the shifting of the bioluminescence spectrum from the reaction of bacterial luciferase by lumazine protein is investigated by methods of fluorescence dynamics. A metastable intermediate is produced on reaction of Vibrio harveyi luciferase with FMNH2 and O2. It has an absorption maximum at 374 nm and a rotational correlation time (phi) derived from the decay of its fluorescence (maximum 500 nm) anisotropy of 90 ns (2 degrees C). Lumazine protein from Photobacterium phosphoreum has an absorption maximum at 417 nm and a fluorescence maximum at 475 nm. Lumazine protein forms a protein-protein complex with luciferase, and the complex has a phi of approximately 100 ns. A mixture of lumazine protein and the intermediate would be expected to have an average correlation time (phi av) around 100 ns, but instead, the result is anomalous. The phi av is much lower and is also wavelength dependent. For excitation at 375 nm, which is mainly absorbed in the flavin chromophore of the intermediate, phi av = 25 ns, but at 415 nm, mainly absorbed by the lumazine derivative ligand of lumazine protein, phi av approximately 50 ns. It is proposed that protein-protein complexation occurs between lumazine protein and the luciferase intermediate and that in this complex energy transfer from the flavin to the lumazine is the predominant channel of anisotropy loss. A distance of 20 A between the donor and acceptor is calculated. In the bioluminescence reaction of intermediate with tetradecanal, a fluorescent transient species is produced which is the bioluminescence emitter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photochemical reaction of 7,8-dihydrorhodopsin at low temperatures

The photoreaction of 9-cis-7,8-dihydrorhodopsin was examined at liquid nitrogen temperatures (-180 degrees C) in order to elucidate the photochemical events in visual pigments. This rhodopsin analog was prepared by incubating 9-cis-7,8-dihydroretinal with bovine opsin in the dark. 9-cis-7,8-Dihydrorhodopsin (lambda max = 427 nm) was cooled to -180 degrees C, and then irradiated at -180 degrees C with a 390 nm light, resulting in formation of its bathochromic product (lambda max = 465 nm). This result indicates that the presence of four double-bonds adjacent to the Schiff base nitrogen is sufficient to allow formation of a bathochromic product. Thus, the mechanism of formation of bathorhodopsin (in bovine rhodopsin system) may be considered as some change of the interaction between the conjugated double-bond system from C-9 to the Schiff base nitrogen and its surrounding charges in opsin, caused by rotation of 11-12 double-bond.     

Raman microscope studies on the primary photochemistry of vertebrate visual pigments with absorption maxima from 430 to 502 nm

Raman microscope vibrational spectra have been recorded from single photoreceptor cells frozen at 77 K. Spectra of photostationary steady-state mixtures of visual pigments and their primary photoproducts were obtained from toad red rods (lambda max 502 nm), angelfish rods (lambda max 500 nm), gecko blue rods (lambda max 467 nm), and bullfrog green rods (lambda max 430 nm). All four photoproducts have enhanced low-wavenumber Raman lines at approximately 850, 875, and 915 cm-1 and show the anomalous decoupling of the 11- and 12-hydrogen out-of-plane (HOOP) wagging vibrations, as is observed in the bovine primary photoproduct. The low-wavenumber lines are enhanced in the resonance Raman spectrum by conformational distortion, and the uncoupling of the 11- and 12-hydrogen wags is caused by additional protein perturbations. The similarity of the HOOP modes in all four photoproducts indicates that the protein perturbations that uncouple the 11- and 12-hydrogen wags and that enhance the HOOP modes are very similar. Thus, these perturbations of the photoproduct Raman spectrum cannot be caused by the same protein-chromophore interactions that are responsible for wavelength regulation in these pigments.     

Mutant of Escherichia coli that instantaneously loses the ability to adsorb lambda bacteriophage upon exposure to high temperature.

lad (lambda adsorption), an Escherichia coli mutant that loses the ability to adsorb lambda phage immediately after a shift to high temperature (e.g., 42 C), was isolated. This property for phage adsorption is irreversible and has been observed with phage lambda and 21 but not with phages 434, phi 170, and phi 80. A crude receptor preparation, extracted from lad cells will cholate-ethylenediaminetetraacetic acid by the procedure of Randall-Hazelbauer and Schwartz (1973), inactivated the phage lambda only at low temperature.     

Specific reaction of 9-cis-retinoyl fluoride with bovine opsin

Opsin readily undergoes Schiff base formation between an active site lysine and 9-cis- or 11-cis-retinaldehyde to form the visual pigments isorhodopsin (lambda max = 487 nm) and rhodopsin (lambda max = 500 nm), respectively (Dratz, 1977). It would be predicted that 9-cis-retinoyl fluoride (1), an isostere of 9-cis-retinal, should be an active site directed, mechanism-based labeling agent of opsin, since a stable peptide bond should be formed instead of a Schiff base. It is shown here that 9-cis-retinoyl fluoride (1) reacts with opsin in a time-dependent fashion (t1/2 = 9 min at 25 microM 1) to form a new, nonbleachable pigment with a lambda max of approximately 365 nm. beta-Ionone competitively slows down the rate of the reaction. The absorbance of the new pigment at approximately 365 nm is similar to that of model amide compounds. This result is consistent in a general and qualitative way with the Nakanishi-Honig point-charge model for visual pigments which requires that the chromophore be charged, a situation not possible when the retinoid is linked to opsin via a peptide bond rather than a protonated Schiff base [Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawinowicz, M.A., Arnabaldi, M., & Motto, M.G. (1979) J. Am. Chem. Soc. 101, 7084-7086]. 9-cis-Retinoyl fluoride (1) is approximately 4-fold more potent than all-trans-retinoyl fluoride (2) as an inactivator of bovine opsin. Importantly, 13-cis-retinoyl fluoride (3) is inactive, and no new absorption band at 365 nm is observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assignment of fingerprint vibrations in the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin: implications for chromophore structure and environment

13C- and 2H-labeled retinal derivatives have been used to assign normal modes in the 1100-1300-cm-1 fingerprint region of the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin. On the basis of the 13C shifts, C8-C9 stretching character is assigned at 1217 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1214 cm-1 in bathorhodopsin. C10-C11 stretching character is localized at 1098 cm-1 in rhodopsin, at 1154 cm-1 in isorhodopsin, and at 1166 cm-1 in bathorhodopsin. C14-C15 stretching character is found at 1190 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1210 cm-1 in bathorhodopsin. C12-C13 stretching character is much more delocalized, but the characteristic coupling with the C14H rock allows us to assign the "C12-C13 stretch" at approximately 1240 cm-1 in rhodopsin, isorhodopsin, and bathorhodopsin. The insensitivity of the C14-C15 stretching mode to N-deuteriation in all three pigments demonstrates that each contains a trans (anti) protonated Schiff base bond. The relatively high frequency of the C10-C11 mode of bathorhodopsin demonstrates that bathorhodopsin is s-trans about the C10-C11 single bond. This provides strong evidence against the model of bathorhodopsin proposed by Liu and Asato [Liu, R., & Asato, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 259], which suggests a C10-C11 s-cis structure. Comparison of the fingerprint modes of rhodopsin (1098, 1190, 1217, and 1239 cm-1) with those of the 11-cis-retinal protonated Schiff base in methanol (1093, 1190, 1217, and 1237 cm-1) shows that the frequencies of the C-C stretching modes are largely unperturbed by protein binding. In particular, the invariance of the C14-C15 stretching mode at 1190 cm-1 does not support the presence of a negative protein charge near C13 in rhodopsin. In contrast, the frequencies of the C8-C9 and C14-C15 stretches of bathorhodopsin and the C10-C11 and C14-C15 stretches of isorhodopsin are significantly altered by protein binding. The implications of these observations for the mechanism of wavelength regulation in visual pigments and energy storage in bathorhodopsin are discussed.     

pH dependence of photolysis intermediates in the photoactivation of rhodopsin mutant E113Q

Glutamic acid at position 113 in bovine rhodopsin ionizes to form the counterion to the protonated Schiff base (PSB), which links the 11-cis-retinylidene chromophore to opsin. Photoactivation of rhodopsin requires both Schiff base deprotonation and neutralization of Glu-113. To better understand the role of electrostatic interactions in receptor photoactivation, absorbance difference spectra were collected at time delays from 30 ns to 690 ms after photolysis of rhodopsin mutant E113Q solubilized in dodecyl maltoside at different pH values at 20 degrees C. The PSB form (pH 5. 5, lambda(max) = 496 nm) and the unprotonated Schiff base form (pH 8. 2, lambda(max) = 384 nm) of E113Q rhodopsin were excited using 477 nm or 355 nm light, respectively. Early photointermediates of both forms of E113Q were qualitatively similar to those of wild-type rhodopsin. In particular, early photoproducts with spectral shifts to longer wavelengths analogous to wild-type bathorhodopsin were seen. In the case of the basic form of E113Q, the absorption maximum of this intermediate was at 408 nm. These results suggest that steric interaction between the retinylidene chromophore and opsin, rather than charge separation, plays the dominant role in energy storage in bathorhodopsin. After lumirhodopsin, instead of deprotonating to form metarhodopsin I(380) on the submillisecond time scale as is the case for wild type, the acidic form of E113Q produced metarhodopsin I(480), which decayed very slowly (exponential lifetime = 12 ms). These results show that Glu-113 must be present for efficient deprotonation of the Schiff base and rapid visual transduction in vertebrate visual pigments.     

Methylation of the active-site lysine of rhodopsin

Purified bovine rhodopsin was reductively methylated with formaldehyde and pyridine/borane with the incorporation of approximately 20 methyl groups in the protein. Rhodopsin contains 10 non-active-site lysines, which account for the uptake of the 20 methyl groups. The permethylated rhodopsin thus formed is active toward bleaching, regeneration with 11-cis-retinal, and the activation of the GTPase (G protein) when photolyzed. The critical active-site lysine of permethylated rhodopsin can be liberated by photolysis. This lysine can be reductively methylated at 4 degrees C. Methylation under these conditions leads to the incorporations of approximately 1.5 methyl groups per opsin molecule using radioactive formaldehyde, with the ratio of epsilon-dimethyllysine:epsilon-monomethyllysine:lysine being approximately 5:4:1. The modified opsin(s) can regenerate with 11-cis-retinal to produce a mixture of active-site methylated and unmethylated rhodopsins having a lambda max = 512 nm. Using [14C]formaldehyde and [3H]retinal followed by reduction of the Schiff base, digestion, and chromatography showed that the active-site N-methyllysine was bound to the retinal. Treatment of the methylated opsin mixture (containing 1.5 active-site methyl groups) with o-phthalaldehyde/mercaptoethanol to functionalize the opsin bearing unreacted lysine, followed by regeneration with 11-cis-retinal and chromatographic separation, led to the preparation of the pure active-site epsilon-lysine monomethylated rhodopsin with a lambda max = 520 nm, significantly shifted bathochromically from rhodopsin or permethylated rhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The quantum efficiency of the bacteriorhodopsin photocycle.

The quantum yield of the primary photoprocess in light-adapted bacteriorhodopsin (phi 1) was determined at room temperature with low-intensity 530 nm neodymium laser excitation, with bovine rhodopsin as a relative actinometer. The observed value of phi 1 - 0.25 +/- 0.05, and the previously determined parameter phi 1/phi 2 - 0.4 [where phi 2 denotes the quantum efficiency of the back photoprecess from the primary species K (590)] imply that phi 1 + phi 2 approximately equal 1. This feature, also characterizing the photochemistry of rhodopsin, bears on the nature and mechanism of the primary event in both systems.     

Absolute absorption spectra of batho- and photorhodopsins at room temperature. Picosecond laser photolysis of rhodopsin in polyacrylamide.

Picosecond laser photolysis of rhodopsin in 15% polyacrylamide gel was performed for estimating absolute absorption spectra of the primary intermediates of cattle rhodopsin (bathorhodopsin and photorhodopsin). Using a rhodopsin digitonin extract embedded in 15% polyacrylamide gel, a precise percentage of bleaching of rhodopsin after excitation of a picosecond laser pulse was measured. Using this value, the absolute absorption spectrum of bathorhodopsin was calculated from the spectral change before and 1 ns after the picosecond laser excitation (corresponding to the difference spectrum between rhodopsin and bathorhodopsin). The absorption spectrum of bathorhodopsin thus obtained displayed a lambda max at 535 nm, which was shorter than that at low temperature (543 nm) and a half band-width broader than that measured at low temperature. The oscillator strength of bathorhodopsin at room temperature was smaller than that at low temperature. The absolute absorption spectrum of photorhodopsin was also estimated from the difference spectrum measured at 15 ps after the excitation of rhodopsin (Shichida, Y., S. Matuoka, and T. Yoshizawa. 1984. Photobiochem. Photobiophys. 7:221-228), assuming a sequential conversion of photorhodopsin to bathorhodopsin. Its lambda max was located at approximately 570 nm, and the oscillator strength was smaller than those of rhodopsin and bathorhodopsin.     

Spatial relationship between a fast-reacting thiol and a reactive lysine residue of myosin subfragment 1

Fluorescence energy transfer was used to examine the spatial proximity between two key side chains in myosin subfragment 1 (S-1), viz., the reactive thiol (SH1) located on the C-terminal 20K tryptic fragment and the reactive lysyl (RLR) on the N-terminal 27K tryptic fragment of S-1 heavy chain. S-1 was specifically labeled at SH1 with an energy donor, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS), and at RLR with an energy acceptor, 2,4,6,-trinitrobenzenesulfonate (TNBS). Prior blocking of SH1 with AEDANS increased the pK of RLR from 9.04 to 9.42. Trinitrophenylation of SH1-blocked S-1 was about 50% slower and sharply reduced the Ca2+ ATPase activity. Reciprocally, blocking of RLR with TNBS slowed the rate of reaction of SH1 and AEDANS by 40-60%. Addition of the second label does not grossly alter the conformation resulting from the first label. S-1 labeled at RLR with TNBS and at SH1 with optically inert iodoacetamide shows the same TNP difference spectrum +/- MgADP (lambda min 365 nm) as S-1 with S 1 free. Also, S-1 labeled at SH1 with AEDANS and at RLR with an optically inert methyl group shows the same AEDANS emission spectrum (lambda em max 475 nm), excited-state lifetime (tau = 20.3 ns) and rotational correlation time (phi = 106 ns) as S-1 with RLR free. When the decrease of either the quantum yield or the excited-state lifetime of the donor in the absence and presence of the acceptor was measured, the energy transfer efficiency was found to be 70%. The apparent interchromophore distance was calculated to be 2.6 nm through the use of the F?rster equation with an uncertainty of less than 12%.