M. A. Ostrovsky

Comparative study of spectral and antioxidant properties of pigments from the eyes of two Mysis relicta (Crustacea, Mysidacea) populations, with different light damage resistance

Abstract Retinal visual and screening pigments of two populations (one marine and the other freshwater) of the opossum shrimp Mysis relicta Lovén (Crustacea, Mysidacea), which have different ocular tolerance to light, was investigated. Visual pigments were extracted by detergent and their bleaching difference spectra were determined. The difference between the visual pigment absorption maximum of the two populations correlated with their difference in spectral sensitivity. Using buffer or neutral methanol, a yellow pigment was extracted which had absorption maxima at 440 nm and 325 nm and bright blue fluorescence (λmax 415 nm). A screening pigment (ommochrome) with maximum at 525 nm was extracted by acid methanol, and was probably related to the group of ommines. The eyes of the lake population had 1.8–2.7 times less of this pigment than the eyes of the sea population. The sea population is more resistant to photo-induced accumulation of thiobarbituric acid-reactive substances in eye tissues. This resistance may be due to the higher ommochrome content.

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.

Role of thermoinduced dissociation in interaction between α-crystallin as an oligomeric chaperone and glyceraldehyde-3-phosphate dehydrogenase as an oligomeric protein substrate

The key function of small heat shock proteins(sHsp), a class of molecular chaperones, is the prevention of aggregation of denatured proteins. The chaperonlike activity of sHsp is determined by their interaction with unfolded protein substrates formed understress conditions (e.g., in stress induced by heat orultraviolet radiation).The proteins of the sHsp family are oligomers consisting of subunits with a molecular weight of 12–42kDa. The molecular weight of sHsp oligomers usuallyvaried in the range from 150 to 800 kDa. The keystructural characteristic of sHsp oligomers is theirability to undergo reversible dissociation at elevatedtemperatures. This yields oligomeric forms of asmaller size, in which the recognition sites for proteinsubstrates open [1–3]. Thus, dissociation of sHsp oligomers may be a necessary conditions for the expression of chaperonlike activity by small heat shock proteins [1–4]. This assumption was confirmed usingHsp26 from

Studies of α- and βL-Crystallin Complex Formation in Solution at 60°C

Studies of molecular mechanisms of chaperone-like activity of α-crystallin became an active field of research over last years. However, fine interactions between α-crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between α- and βL-crystallins was studied during thermal denaturation of βL-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of α- and βL-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration ∼14 nm, mean molecular mass ∼4 MDa and maximal size over 40 nm. In pure βL-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the α-crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of βL-crystallin it binds with modified α-crystallin or, alternatively, βL-crystallin complexation and α-crystallin modifications are concurrent. Estimates of the α-βL-crystallin complex size and relative contents of α- and α-βL-crystallins in the complex suggest that several α-crystallin molecules are involved in complex formation.

Comparison of photosensitizing effect of lipofuscin granules from retinal pigment epithelium of human donor eyes and their fluorophore A2E.

Some forms of macular degeneration of retina, such as Schtargardt’s disease or age-related macular degeneration of retina, are characterized by the accumulation of a great number of lipofuscin granules in RPE cells, which precedes RPE atrophy and degeneration of photoreceptors. The accumulation of these granules apparently accounts for the aggravating effect of light in these severe eye diseases [1]. It was believed earlier that lipofuscin granules represent harmless ballast accumulated in postmitotic cells with aging. However, it was shown that these granules are light-sensitive and, under exposure to visible light, are able to generate toxic reactive oxygen species (singlet oxygen, superoxide radicals, and hydroperoxides) [2–4].

Heat-induced Complex Formation in Solutions of α- and βL-Crystallins: A Small-Angle X-ray Scattering Study

To gain more insight into molecular mechanisms of chaperone-like activity of α -crystallin, the effect of heating on the protein particle size in solutions of α and β L -crystallins was studied by small-angle X-ray scattering (SAXS). Heating a mixture of α - and β L -crystallins in solution at 60 ° C resulted in the formation of several populations of high-molecular-weight complexes. In contrast, no complex formation was observed at 60 ° C in solutions of β L -crystallin alone. The data obtained provided evidence that α -crystallin at 60 ° C underwent rearrangements that roughly doubled its molecular weight. Based on the results of size estimation for complexes of α - and β L -crystallins, it was concluded that they were assembled via interaction between several macromolecules of α -crystallin. Crystallins are eye lens proteins. α -Crystallin, like heat-shock proteins, functions as a molecular chaperone in preventing aggregation of proteins in solution, induced by heating, UV irradiation, or other denaturing factors [1‐10]. α -Crystallin forms complexes with partially denatured proteins and thereby prevents their aggregation and opacification of the solution. With age, complexes of α -crystallin with other crystallins accumulate in the lens [4]. Chaperone-like activity of α -crystallin is thought to play an important role in maintaining transparency of the eye lens throughout life. Loss of this property by α -crystallin is supposed to contribute to the formation of senile cataract. The study of molecular mechanisms of chaperone-like activity of α -crystallin and especially of the structure of its complexes with denatured proteins is an important physiological problem. Structural information about the complexes can be obtained using SAXS [11]. In this study, we report the results of SAXS analysis of the complexes of α -crystallin with proteins subjected to denaturing treatment.

Natural Dipeptides as Mini-Chaperones: Molecular Mechanism of Inhibition of Lens βL-Crystallin Aggregation

The effect of histidine-containing dipeptides-carnosine and N-acetylcarnosine-on preventing and treating of cataracts of various etiologic origins has been demonstrated in many studies in vivo, while the precise molecular mechanism of their action is actually obscure. Cataract has been recently attributed to conformational diseases due to the association of lens structure protein aggregation with cataract pathogenesis. In our study, effect of histidine-containing dipeptides-carnosine, N-acetylcarnosine, and anserine-on the UV induced βL-crystallin aggregation was studied in vitro. It was first demonstrated that N-acetylcarnosine and anserine (10-40 mM) considerably suppressed UV induced aggregation of βL-crystallin, while carnosine exerted no effect. Positive correlation between anti-aggregating activity of the compounds used and their hydrophobicity was obtained. It was revealed that N-acetylcarnosine and anserine inhibited the initial stages of the protein photochemical damage. A decrease in the size of protein aggregates was detected in the presence of N-acetylcarnosine and anserine. UV irradiation of βL-crystallin resulted in a significant increase in the number of protein carbonyl groups, and the dipeptides studied did not affect this process. We suppose that N-acetylcarnosine and anserine inhibit βL-crystallin aggregation via formation of a protein-dipeptide complex that prevents macromolecular conformational changes and ensuing protein aggregation.

Estimation of fluorescence lifetime of lipofuscin fluorophores contained in lipofuscin granules of retinal pigment epithelium of human cadaver eyes without signs of pathology

The fluorescence lifetimes of lipofuscin fluorophores contained in chloroform extracts from retinal pigment epithelium (RPE) of human cadaver eyes without signs of pathology were evaluated by single photon counting. The comparison of fluorescence lifetimes of N-retinylidene-N-retinylethanolamine (A2E) and its photooxidation and photodegradation products has been carried out. It was shown that the contribution of A2E to the total fluorescence of chloroform extract from lipofuscin granules is not major. The results are important for the improvement of noninvasive diagnostic method of degenerative diseases of the retina and RPE—fundus autofluorescence (FAF).

Nonreciprocal XeCl laser-induced aggregation of β-crystallins in water solution

The aggregation of a β-crystallin water solution exposed to XeCl laser radiation demonstrates the dependence of scattering-exposure curve (scattering versus exposure) on laser intensity. The main features of this dependence can be understood by the relaxation of a partly denaturated state of a protein within some finite relaxation time. These photoactivated states originate from the absorption of UV photons. Two partly denaturated (photoactivated) monomers, as well as other aggregates, can aggregate, giving rise to sharply increasing probe light scattering after some lag time of irradiation.

Coherent processes in formation of primary products of rhodopsin photolysis

194 The absorption of the light quantum by rhodopsin (R 498 ), a visual pigment, results in the cis → trans isomerization of its chromophore, 11cis -retinal, over approximately 200 fs [1, 2] with a quantum yield of 0.65 [3]. The physical meaning of this fast and efficient photoreaction is in the maximal utilization of the absorbed light quantum energy for isomerization of 11cis -retinal rather than its dissipation into heat or loss as fluorescence. The isomerization of 11cis -retinal leads to conformational rearrangement in the protein moiety of rhodopsin molecule; as a result, rhodopsin as a G-protein binding receptor acquires the ability to interact with G-protein (transducin), thereby initiating phototransduction in the photoreceptor cell. The dynamics of the 11cis -retinal photoisomerization in rhodopsin has been studied by laser kinetic spectroscopy with a femtosecond resolution [1, 2, 4–6]. It has been demonstrated that the first reaction product, photorhodopsin (photo 570 ), is formed over 200 fs to transform into the next product, bathorhodopsin (batho 535 ), in 2–3 ps [1, 4, 5]. The oscillations of the absorption signals of photo 570 , batho 535 , and initial state R 498 , observed during several picoseconds after excitation, have been also discovered. A high rate and quantum yield of 11cis -retinal photoisomerization in rhodopsin as well as the oscillations of absorption signals of the reaction products suggested that this is a coherent reaction. This means that the photoreaction proceeds via passing of the system through nonstationary oscillation states. In terms of the model of two states [1, 7], when two potential energy surfaces (PESs) are involved in photochemical reaction of rhodopsin (Fig. 1), the corresponding processes can be represented as follows. The action of a femtosecond excitation pulse results in a coherent population of certain oscillatory states in the electron-excited molecule, the set of which is named a coherent oscillatory wave packet (hereinafter, wave packet). The coherent photoreaction can be represented as the movement of wave packet over PES S 1 of the excited state along the reaction coordinate. The reaction products are formed Coherent Processes in Formation of Primary Products of Rhodopsin Photolysis