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1.
RESOLUTION OF SPECTRAL AND LIFETIME HETEROGENEITY OF TRYPTOPHAN FLUORESCENCE IN BACTERIORHODOPSIN 总被引:1,自引:0,他引:1
Abstract— The picosecond time-resolved fluorescence decay of bacteriorhodopsin (BR) was analyzed by the maximum entropy method. Results showed five distributions of lifetimes indicating at least five decay components. A wavelength-dependent study of emission decay of BR was carried out in the wavelength region from 310 to 390 nm. The decay at each wavelength was resolvable into four decay components by the discrete exponential analysis. The three short lifetime components (100 ± 20 ps, 400 ± 50 ps and 1.0 ± 0.1 ns) were independent of wavelength, whereas the longest lifetime component was wavelength dependent (varying from 4.1 ns at 310 nm to 5.7 ns at 390 nm). These results are inconsistent with the existing model of associating the fluorescence of bacteriorhodopsin with two or four lifetime components. An attempt is made to associate the five decay components with the emitting tryptophans of BR. 相似文献
2.
Abstract— The photoreaction of the acidified form of bacteriorhodopsin and its 9-cis derivative was studied by low temperature spectroscopy.
A short exposure of the acidified form of bacteriorhodopsin, which was prepared by adding 2 m M HC1 to purple membrane suspension in 67% glycerol at 0°C, to red light at – 72°C resulted in the blue-shift of the spectrum. The feature of the shift was very similar to that accompanied by the formation of stable 9- cis acidified form of bacteriorhodopsin at 0°C, but only 13- cis - and all- trans -retinals were found in the extract from this product. No blue-shifted product was found on irradiation at – 190°C.
Irradiation of the 9- cis form of acidified bacteriorhodopsin at -72°C with blue light caused the isomerization of its 9- cis -retinylidene chromophore to 13- cis and all- trans forms without a significant spectral change. It became greater only after the sample was warmed above – 24°C. These results indicate the presence of the light-induced product which has trans configuration on the 9-10 double bond and exhibits the 9- cis type spectrum. 相似文献
A short exposure of the acidified form of bacteriorhodopsin, which was prepared by adding 2 m M HC1 to purple membrane suspension in 67% glycerol at 0°C, to red light at – 72°C resulted in the blue-shift of the spectrum. The feature of the shift was very similar to that accompanied by the formation of stable 9- cis acidified form of bacteriorhodopsin at 0°C, but only 13- cis - and all- trans -retinals were found in the extract from this product. No blue-shifted product was found on irradiation at – 190°C.
Irradiation of the 9- cis form of acidified bacteriorhodopsin at -72°C with blue light caused the isomerization of its 9- cis -retinylidene chromophore to 13- cis and all- trans forms without a significant spectral change. It became greater only after the sample was warmed above – 24°C. These results indicate the presence of the light-induced product which has trans configuration on the 9-10 double bond and exhibits the 9- cis type spectrum. 相似文献
3.
Abstract— An unusual photo-reaction of benz(a)pyrene has been investigated by measuring prompt and delayed fluorescence spectra. The proposed mechanism involves adsorption of triplet benz(a)pyrene by the polymer and its reaction to give substitution products. Further work is in progress with pyrene, which also undergoes photo-reaction in the presence of polymer. 相似文献
4.
Abstract— The sequence of primary events following light absorption by light adapted bacteriorhodopsin (bR570 ) is considered by analyzing recent picosecond absorption and emission data. The analysis is facilitated by theoretical calculations which allow us to characterize the properties of the first excited singlet state. It is concluded that excitation leads to the eventual population of a photochemically important nonfluorescent excited state (I) which decays into a photoproduct (J625 )- In J625 , which is most probably a ground state molecule, the chromophore has undergone a structural change, presumably trans → 13- cis isomerization. It is suggested that the subsequent process
reflects a relaxation of the protein environment involving proton transfer. 相似文献
reflects a relaxation of the protein environment involving proton transfer. 相似文献
5.
Abstract— Halobacterium halobium cells grown in nicotine-containing medium synthesize bacterio-opsin (bO) but little bacteriorhod-opsin (bR) because nicotine inhibits retinal synthesis. In nicotine-grown cells, bacterio-opsin is found in a specific cell membrane fraction, the brown membrane (b.m.), which consists mainly of small round sheets. Freeze-fracture of isolated brown membrane reveals a dense particle population on its cytoplasmic fracture face, with few particles on the external face. There is no apparent order in the particle distribution and the fracture faces are practically indistinguishable from those of red membrane (r.m.). The absorbance spectrum of b.m. shows peaks at 550 and 410nm, the circular dichroism (CD) spectrum a positive band around 540nm and positive and negative bands around 410nm with a cross-over at 412nm. The photoreaction cycle of bR in b.m. has the same intermediates found in purple membrane (p.m.), but the apparent kinetics resemble those of bR monomers. Addition of 13-cis-, or all-trans-retinal to b.m. induces a rapid 5- to 10-fold increase in the visible absorbance band, and the absorbance maximum shifts to 560nm in the dark. Kinetic analysis of the absorbance increase shows three first order kinetic constants with t½= 0.5 min, 6 min and 100 min, with most of the increase contributed by the fastest component. The CD bilobal pattern typical for purple membrane appears together with the strong negative band at 320nm. Ellipticity change at 590nm is nearly as rapid as the absorbance increase; within 5 min, 85% of the negative band is formed. Electron microscopy after addition of retinal reveals structurally distinct domains in the b.m. sheets which have the characteristic appearance of p.m. However, X-ray reflections are more diffuse compared to p.m. Flash spectroscopy of reconstituted b.m. detects the same photocycle intermediates as in b.m. or p.m., but with apparent kinetics closer to those of purple membrane than before reconstitution. These results show that bO in the b.m. binds retinal to form bR which spontaneously aggregates into a lattice. However, the reconstituted bR lattice is not as well-ordered as in p.m. and some bR is still present in the form of monomers and/or small aggregates. 相似文献
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7.
Abstract— The pulsed laser photolysis of light-adapted bacteriorhodopsin (BR570) is carried out over the temperature range between 25°C and—92°C in neutral and alkaline water-glycerol solutions. The results arc indicative of considerable complexity, introduced by two temperature dependent branching reactions associated with the intermediates K610, L550 and M412, of the BR570 photocycle. (a) At relatively low temperatures the primary photoproduct K-610 equilibrates with a blue-shifted species, Kp. Both K610 and the new intermediate subsequently decay into another species, K'r, in a process which competes with the formation of L550. Finally, K'p converts very slowly to L550. This branched pathway delays the formation of L550 and thus of M412, without affecting the final yield of either species, (b) A thermal back-reaction regenerating BR570 takes place at the stage of L550, inhibiting the formation of M412. The reaction which also predominates at low temperatures, is relatively inefficient at high pH when the forward L550→ M412 step is highly catalyzed. It is the superposition of both branching mechanisms, (a) and (b), which accounts for the complex effects of temperature and pH on the photo-cycle of BR570. Mechanism (b) is accounted for by a molecular scheme in which deprotonation of a tyrosine moiety at the stage of L550 constitutes a prerequisite for deprotonation of the retinal-lysine schiff-base as required for forming M412. This scheme appears to be directly related to the proton pump. Mechanism (a) introduces additional complexity in the photocycle at low temperatures but its molecular aspects are still unclear. 相似文献
8.
Abstract— Some photochemical reactions of bacteriorhodopsin (BR) embedded in NaCl pellets (BR-NaCI) in the visible region are described here. BR in these preparations is a mixture of two classes of species: a drastically blue-shifted form and the unchanged purple pigment. Depending on the illumination history of the BR before being immobilized, both kinds of BR could be demonstrated in light-adapted (LA) and dark-adapted (DA) forms, but light adaptation was not possible once the pellets were made. Analogously to BR suspensions, the light-adapted blue-shifted BRexhibited an a/l-trans type photocycle, but the thermal steps were greatly slowed down (time constants 1 to 5 min). The parent species absorb at 506 nm. The DA blue-shifted BR exhibited absorption changes resolved into two photoreactions, one all-(rans- like (as in LA-BR) and another, 13-cw like, whose decay rate is also greatly slowed down (recovery time several hours). The parent species of the 13-cis like cycle absorb at 480 nm. That pigment fraction in the pellets whose absorption was not blue-shifted, also exhibited similar photoreactions to BR in suspension, but with an overall turnover rate only one order of magnitude slower. From a previous report (Lazarev and Terpugov, Biochim. Biophys. Acta 590 ,324–338,1980) and this one, it appears that the very slow photocycles in NaCl-BR of low moisture content originate from blue-shifted chromophores rather than from unchanged BR. 相似文献
9.
ENERGY STORAGE IN THE PRIMARY PHOTOREACTION OF BOVINE RHODOPSIN. A PHOTOACOUSTIC STUDY 总被引:1,自引:0,他引:1
Abstract— Photoacoustic spectroscopy is used to investigate the uptake of energy in the primary photoreaction of bovine visual pigment (Rhodopsin → Bathorhodopsin). It is shown that very concentrated dried rod outer segment membranes have a sufficient thermal diffusivity to be analyzed by this technique. From the photoacoustic and absorption spectra of these membranes, the low temperature dissipation spectrum has been obtained and the results are consistent with the storage of 145 kJ mol-1 in the primary event of vision having a quantum yield of 0.67. By photoacoustic spectroscopy, this process is continuously monitored from 350 to 550 nm and its efficiency is found to vary by less than 10%, even in the spectral region of the β-band of rhodopsin. 相似文献
10.
Abstract -The rate of formation of the M intermediate ( k M ) in the photocycles of bacteriorhodopsin (bR570 ) and of nitrated bacteriorhodopsin (bR532 n ), is measured over the range between pH 6.5 and 11.5. In the case of bR570 , k M is markedly pH dependent, exhibiting a titration-like curve with pK ∽ 10.3. The pH dependency is completely eliminated by nitration. On the basis of previous work by Lemke and Oesterhelt (1981), the effect is attributed to the specific modification of the Tyr 26 residue. The data are rationalized by a mechanism in which deprotonation of Tyr 26 at the stage of the L intermediate constitutes a prerequisite for deprotonation of the retinal-lysine SchifT base. Both reactions are intimately associated with the photo-induced proton pump mechanism. 相似文献
11.
Abstract— Suspensions of purple membrane fragments showed obvious signs of degradation after illumination with intense pulses of light from 10 ns frequency doubled Nd: YAG laser at 532 nm with intensity densities in excess of 1 MW/cm2 . Using controlled illumination, a small fraction of the bacteriorhodopsin protein molecules were randomly destroyed in samples with a low salt concentration (12.5 m M ) and pH = 7.9. Calculations using information from the changes in the optical absorbance spectrum and transient changes in the optical absorbance spectrum during the photocycle support a model where one protein molecule of the bacteriorhodopsin trimer is photodestroyed, the other two protein molecules switch to a blue state . In the blue state , the protein molecules have a red shifted absorption, with a peak near 600 nm. The blue state molecules show transient absorption changes at 656 nm that are similar to the native bacteriorhodopsin, except the O state is missing or altered. Additionally, the changes in curvature of the purple membrane fragments that occur during the photocycle of intact protein molecules are severely depressed. The addition of salts to the photodestroyed suspension can change the blue state molecules back to a state with an absorption maximum at 568 nm. The salt ions probably shield the other members of the trimer from the photodestroyed protein. In these reconstituted samples, the O state is observed at 656 nm; however, the membrane bending is not observed. 相似文献
12.
I. Gomez R. PICOREL J. M. RAMIREZ R. PEREZ F. F.delCAMPOt † 《Photochemistry and photobiology》1982,35(3):399-403
Abstract— Reversible oxidation of bacteriochlorophyll in two independent mutants of Rhodospirillum rubrum lacking photoreaction center has been studied to obtain further information about the antenna system of that bacterium. Oxidation of antenna bacteriochlorophyll with ferricyanide is accompanied by three distinct absorbance changes in the near infrared: the appearance of a new band at 1230 nm (B1230) and the bleaching of two bands, a major one at 882 nm (B882) and a minor one at 888 nm (B888). The three absorbance changes take place at potentials higher than those required for complete oxidation of photoreaction-center bacteriochlorophyll in wild-type strains. On the other hand, the B882 bleaching and the B1230 appearance occur under similar redox conditions. The above data suggest, first, that in Rds. rubrum , contrary to the current generalized idea, antenna bacteriochlorophyll exhibits at least two absorption bands, B888 and B882; second, that B882 and B1230 correspond to the same bacteriochlorophyll component. In addition, the appearance of B1230 may indicate that antenna bacteriochlorophyll can be partially arranged in an oligomeric fashion, for bands in that spectral region are not exhibited by oxidized monomeric bacteriochlorophyll. 相似文献
13.
S. Druckmann R. Renthal M. Ottolenghi W. Stoeckenius 《Photochemistry and photobiology》1984,40(5):647-651
Abstract —Nanosecond electron beam pulses cause radiolytic reduction of the retinal Schiff base in bacteriorhodopsin. The effects of different scavengers show that both H atoms and the formate radical anion can act as the reducing species. Model system studies on the radiolysis of retinal indicate transient formation of a retinal radical anion and suggest an analogous two-step reaction in bR. Delocalization of the radical in the polyene system and reaction with surrounding groups would account for the relatively low yield of reduced Schiff base and the appearance of intra- and intermolecular crosslinks in irradiated bacteriorhodopsin. 相似文献
14.
用不锈钢/电沉积紫膜薄膜/含水胶(电介质)/钢型菌紫质光电池研究了作用于N-端表面(紫膜外表面)的钾、镁、镧离子对菌紫质光电响应的影响。在所测条件下光电极性均与质子泵方向一致。光电压幅值是离子浓度依赖性的,对钾、镁、镧离子均存在极大值浓度,它们之间有数量级的差异;依赖性曲线趋势和极大值浓度均各自分别与溶液中菌紫质的质子泵效率对阳离子浓度的这种依赖性相一致。不过,光电响应对作用于C-端表面的这些离子不存在上述的浓度依赖性。上述的两种依赖性成相关性说明:电沉积紫膜薄膜中菌紫质的光电响应主要来自质子泵运而非非质子离子的贡献;存在一合适的离子浓度使菌紫质的光电响应有极大值。用质子泵结构域模型和扩散双电层理论对这种依赖性进行的讨论认为:金属阳离子的结合与屏蔽两种效应可以解释这种依赖性。 相似文献
15.
SPECTRAL EVIDENCE FOR PHOTO-INDUCED ISOMERIZATION OF CAROTENOIDS IN BACTERIAL PHOTOREACTION CENTER 总被引:1,自引:0,他引:1
Abstract The aim of this work was to determine whether spirilloxanthin and sphaeroidene bound to the same site of the photoreaction center isolated from Rhodospirillum rubrum can display cis–trans isomerization under conditions that lead to formation of triplet state PR of the primary electron donor. To this end, we monitored changes in the absorption spectrum of these bound carotenoids as induced by red light at low redox potentials. This experiment was performed both with the intact photoreaction center isolated from strain Sl and with the photoreaction center isolated from carotenoidless strain G9 reconstituted with either spirilloxanthin or sphaeroidene. In both preparations, spirilloxanthin exhibited light-induced absorption changes that can be interpreted as a cis–trans isomerization. Under our experimental conditions, the absorption changes attained their full extent in about 1 min and were not or were only partially reversed when the light was switched off. Under our experimental conditions, the extent of these changes indicate that about 15% of the bound spirilloxanthin undergoes isomerization. Sphaeroidene artificially attached to the G9 photoreaction center also undergoes light-induced absorbance changes, but these cannot easily be interpreted as a cis–trans isomerization. 相似文献
16.
We have used a signal averaging laser (30 ps pulse)-streak camera system to measure fluorescence anisotropy decays of dyes in solution and protein tryptophan chromophores with picosecond time resolution. Emission polarization as a function of time can be measured directly and simultaneously for both polarization directions and stored in an optical multichannel analyzer. The corrected anisotropy is computed after background subtraction. Rotations of free dyes in solution (Sulforhodamine 101, tryptophan) and the temperature-dependent internal motions of human serum albumin (tryptophan emission) are displayed. 相似文献
17.
18.
Chung-Lu Hsieh M. A. El-Sayed Malcolm Nicol Mark Nagumo † Jai-Hyung Lee † 《Photochemistry and photobiology》1983,38(1):83-94
Abstract— Resonance Raman spectra of the picosecond bacteriorhodopsin intermediate(s) have been obtained by microbeam, flow and subtraction techniques using a synchronously pumped, cavity-dumped dye laser. Nanosecond spectra also were measured with this laser by cavity dumping without mode-locking. The picosecond spectra in the fingerprint region, which is sensitive to the configuration of the retinal chromophore, differ from spectra of the parent bR570 but could be correlated to the spectrum of bRDA550 , a “13-cis” species which has been determined from spectra of bR570 and bRDA560. The picosecond transient and bRDA550 also are similar in the 950–1050 cm-1“deuteration fingerprint” region when the medium is changed from H2O to D2O. These results suggest that trans—cis isomerization occurs during the 40-ps pulse duration. The shift relative to the parent bR570 in the ethylenic stretch region suggests that the picosecond and nanosecond transients absorb at wavelengths longer than 570 nm. The C band at 1646 cm-1 is found to shift or to broaden upon photolysis in the picosecond time scale. This might suggest a change in the electronic structure of the group and its environment on the picosecond time domain. The nanosecond spectra obtained in this work (with 15-ns pulses) are similar to the spectra previously observed on the 100-ns time scale but are slightly different from the picosecond spectrum. These data suggest that more than one transient species appears on the picosecond-to-nanosecond time scale. The temporal evolution of Raman bands in the fingerprint as well as the low energy (950–1050 cm-1) region and its implications are discussed. 相似文献
19.
Abstract A mode-locked Nd:YAG laser was used to excite the aromatic amino acid residues of bacteriorhodopsin in the purple membrane and the tryptophan (Trp) fluorescence decay analyzed with a streak camera (λ> 380 nm). The decay kinetics are resolvable into two first-order half-times (1.5 and 0.17 ns, respectively), while for retinylidene-free bacterioopsin, only the longer-lived Trp emission was observed. The shorter-lived species reappeared upon regeneration of bacteriorhodopsin by addition of retinal to bacterioopsin but not on treatment of the latter with an equivalent of retinol. It is proposed that these results are consistent with a structural model in which the 7-8 Trp's distributed among sections A, C, E and F of the seven helical segments A-G of native bacteriorhodopsin are distinguishable by their distances from the chromophore. Assuming a Förster mechanism for energy transfer with Ro = 25 and 32 Å, respectively, for retinylidene chromophore and retinol the Trp's may be divided into two groups: (i) those completely quenched by retinol and partly quenched by retinal (τ= 0.17) with R ≃ 18 Å and (ii) those (τ= 1.5 ns) which are quenched neither by chromophore nor retinol with R > ca. 30 Å. These results are consistent with and support some of the best models of Engelman et al. (1980) for the protein conformation in the purple membrane. 相似文献
20.
A. Yu. Sharonov N. V. Tkachenko V. V. Savransky A. K. Dioumaev 《Photochemistry and photobiology》1991,54(6):889-895
Abstract– The kinetics of the absorption changes associated with the perturbation of aromatic acids during the photocycle of bacteriorhodopsin (bR) were studied at room temperature with microsecond time-resolution. Flash experiments with nanosecond excitation at 532 nm were performed on the purple membrane suspension at a number of measuring wavelengths in the spectral range250–630 nm (to monitor both non-chromophore changes and the photocycle kinetics). The kinetic data collected at different wavelengths were simultaneously fitted with a sum of exponentials to obtain time-resolved UV-VIS difference spectra of photocycle intermediates. This approach allowed us to separate kinetically distinct contributions coupled with tryptophan(s) and tyrosine(s) perturbations. Contributions associated with a reversible perturbation of tryptophans appeared with complex (multistep) kinetics during the bRM transitions and relaxed in a single step during the M0 transition. A contribution associated with perturbation of the local environment of tyrosine appeared before the L and relaxed during the Ob̊ transition. 相似文献