Mikhail A. Ostrovsky

A new hybrid protein for production of recombinant bacteriorhodopsin in Escherichia coli

Unique properties of bacteriorhodopsin, namely, photochromism and high thermal stability, make this protein an attractive target for physico-chemical studies, as well as for various biotechnological applications. Using Mistic as a suitable carrier for insertion of recombinant membrane proteins into cytoplasmic membrane of Escherichia coli, we developed a system for overexpression of bacteriorhodopsin and worked out an efficient procedure for its purification and renaturation with the final yield of 120 mg/l of refolded protein, which is the highest value reported to date for bacteriorhodopsin produced in E. coli. Functional activity of recombinant bacteriorhodopsin was confirmed by spectroscopic and electrochemical assays.

Eye Adaptation to Different Light Environments in Two Populations of Mysis relicta: A Comparative Study of Carotenoids and Retinoids

Abstract The content of carotenoids and retinoids was compared in the eyes of two Finnish populations of the opossum shrimp, Mysis relicta, which have been reproductively isolated for at least 9000 years: one from the deep, dark, Lake Pääjärvi, the other from the Baltic Sea (Pojoviken Bay). The eyes of the lake population (LP) are highly susceptible to light damage, while those of the sea population (SP) are more resistant. Carotenoids are known to act as antioxidants protecting cells against free radicals and reactive oxygen species; retinoids, on the contrary, may be phototoxic in certain conditions. Analyzed by spectrophotometry and HPLC, the carotenoid content was broadly similar in the eyes of the two populations as regards both total amount and relative proportions of more than 20 components. Noteworthy differences were found in only three of the major components, among these astaxanthin, which was two times higher in SP compared with LP. The most interesting finding was the 1.6-fold higher content of retinoids in LP compared with SP, with retinol as the dominant component (40% of total) in both populations. Retinol is a precursor of the visual-pigment chromophore retinal. The result is consistent with the idea that animals inhabiting extremely dim light environments, where very little photoregeneration of metarhodopsin to rhodopsin can occur, need a large store of chromophore (or precursors) for effective “dark” regeneration of visual pigment. We suggest that almost all the rhodopsin is then in the native state and massive pigment activation following exposure to stronger light may trigger photoreceptor damage. If such animals are handled without due light protection, e.g., when transferred to a new habitat or collected for biological experiments, their vision will be severely impaired.

Ionochromic properties of long-wave-sensitive cones in the goldfish retina: an electrophysiological and microspectrophotometric study

Long-wave-sensitive (LWS) cone visual pigments are sensitive to the concentration of chloride ions and show a spectral shift to shorter wavelengths when exposed to low chloride levels. We have used the aspartate-isolated mass receptor potential of the electroretinogram (ERG) to establish whether the spectrally shifted cone pigment is functional. In the goldfish, Carassius auratus, the lambda(max) of the LWS porphyropsin is displaced from about 622 nm to around 606 nm when chloride is replaced by gluconate. The electrical response of the LWS cones (but not MWS cones and rods) is selectively and reversibly abolished by the removal of chloride ions. The effect is concentration dependent and a decrease to half the normal chloride ion concentration is sufficient to cause a decrease in the response.

Chloride conducting light activated channel GtACR2 can produce both cessation of firing and generation of action potentials in cortical neurons in response to light

Optogenetics is a powerful technique in neuroscience that provided a great success in studying the brain functions during the last decade. Progress of optogenetics crucially depends on development of new molecular tools. Light-activated cation-conducting channelrhodopsin2 was widely used for excitation of cells since the emergence of optogenetics. In 2015 a family of natural light activated chloride channels GtACR was identified which appeared to be a very promising tool for using in optogenetics experiments as a cell silencer. Here we examined properties of GtACR2 channel expressed in the rat layer 2/3 pyramidal neurons by means of in utero electroporation. We have found that despite strong inhibition the light stimulation of GtACR2-positive neurons can surprisingly lead to generation of action potentials, presumably initiated in the axonal terminals. Thus, when using the GtACR2 in optogenetics experiments, its ability to induce action potentials should be taken into account. Our results also open an interesting possibility of using the GtACR2 both as cell silencer and cell activator in the same experiment varying the pattern of light stimulation.

Lake and Sea Populations of Mysis relicta (Crustacea,Mysida) with Different Visual-Pigment Absorbance Spectra Use the Same A1 Chromophore

Glacial-relict species of the genus Mysis (opossum shrimps) inhabiting both fresh-water lakes and brackish sea waters in northern Europe show a consistent lake/sea dichotomy in eye spectral sensitivity. The absorbance peak (λmax) recorded by microspectrophotometry in isolated rhabdoms is invariably 20–30 nm red-shifted in “lake” compared with “sea” populations. The dichotomy holds across species, major opsin lineages and light environments. Chromophore exchange from A1 to A2 (retinal → 3,4-didehydroretinal) is a well-known mechanism for red-shifting visual pigments depending on environmental conditions or stages of life history, present not only in fishes and amphibians, but in some crustaceans as well. We tested the hypothesis that the lake/sea dichotomy in Mysis is due to the use of different chromophores, focussing on two populations of M. relicta from, respectively, a Finnish lake and the Baltic Sea. They are genetically very similar, having been separated for less than 10 kyr, and their rhabdoms show a typical lake/sea difference in λmax (554 nm vs. 529 nm). Gene sequencing has revealed no differences translating into amino acid substitutions in the transmembrane parts of their opsins. We determined the chromophore identity (A1 or A2) in the eyes of these two populations by HPLC, using as standards pure chromophores A1 and A2 as well as extracts from bovine (A1) and goldfish (A2) retinas. We found that the visual-pigment chromophore in both populations is A1 exclusively. Thus the spectral difference between these two populations of M. relicta is not due to the use of different chromophores. We argue that this conclusion is likely to hold for all populations of M. relicta as well as its European sibling species.

Conical intersection participation in femtosecond dynamics of visual pigment rhodopsin chromophore cis-trans photoisomerization

242 The first and the only photochemical reaction in vision is the photoisomerization of 111cisretinal, the chromophore of the visual pigment rhodopsin, to the trans form. This reaction is one of the most rapid and effective responses in nature. In rhodopsin, it proceeds faster than for 200 fs [1–3], with a quantum yield of 0.65 [4], and is accompanied by a significant energy storage (~35 kcal/mol) [5]. It should be noted that, in a solution, free retinal undergoes photoisomerization within several picoseconds [6] with a quantum yield of 0.15 [7]. The acceleration of isomerization of 111cis retinal in the chromophore site of rhodopsin and the increase in the quantum yield of the protein are pree sumably associated with the influence of the protein environment on the chromophore. The elementary primary act of retinal isomerizaa tion in rhodopsin is interpreted as a nonactivated energy passage through the conical intersection of the potential energy surfaces (PESs) of the electronically excited state S 1 and the ground state of the product S 0 [8, 9]. The observed lowwfrequency oscillations in the kinetic curves of difference absorption of photorr hodopsin, the primary product of retinal photoii somerization in rhodopsin, suggest that the passage of the S 1 /S 0 PES conical intersection is accompanied by the transfer of the coherent vibrational wave packet on the PES of the product [1, 3, 10]. A direct experimental detection of such an event as the passage through a conical intersection and the determination of the time required to reach it from the initial Franck–Condon (FC) state, which forms as a result of excitation with a femtosecond pulse, is a nonn trivial task. Recently, a group of American [8] and Japan [11] authors obtained experimental evidence for the existence of a conical intersection in the reaction of retinal photoisomerization in rhodopsin. It was shown that the time required to reach the conical intersection point is about 80 fs for rhodopsin of both vertebrates [8] and invertebrates [11]. In contrast to the reaction of retinal photoisomerization in rhodopp sin, is has recently been shown for retinal in solution that the conical intersection is reached within 2.9– 3.8 ps [12]. The time required to reach the conical intersection [12] was determined by studying the kinetics of stimulated emission of retinal in solution. In this work, the difference spectra and kinetics of the rhodopsin photoinduced absorption and the anisotropy of the …

Femtosecond Laser Spectroscopy of the Rhodopsin Photochromic Reaction: A Concept for Ultrafast Optical Molecular Switch Creation (Ultrafast Reversible Photoreaction of Rhodopsin)

Ultrafast reverse photoreaction of visual pigment rhodopsin in the femtosecond time range at room temperature is demonstrated. Femtosecond two-pump probe experiments with a time resolution of 25 fs have been performed. The first pump pulse at 500 nm initiated cis-trans photoisomerization of rhodopsin chromophore, 11-cis retinal, which resulted in the formation of the primary ground-state photoproduct within a mere 200 fs. The second pump pulse at 620 nm with a varying delay of 200 to 3750 fs relative to the first pump pulse, initiated the reverse phototransition of the primary photoproduct to rhodopsin. The results of this photoconversion have been observed on the differential spectra obtained after the action of two pump pulses at a time delay of 100 ps. It was found that optical density decreased at 560 nm in the spectral region of bathorhodopsin absorption and increased at 480 nm, where rhodopsin absorbs. Rhodopsin photoswitching efficiency shows oscillations as a function of the time delay between two pump pulses. The quantum yield of reverse photoreaction initiated by the second pump pulse falls within the range 15% ± 1%. The molecular mechanism of the ultrafast reversible photoreaction of visual pigment rhodopsin may be used as a concept for the development of an ultrafast optical molecular switch.

The 100th Anniversary of the Russian Pavlov Physiological Society

The first All-Russian Congress of Physiologists was held in Petrograd (Saint Petersburg), Russia in April 1917 at the Medical Institute for Women ([11][1]). Ivan Pavlov was the chairman of the organizing committee for this first “Sechenov Physiological Congress,” but he was unable to attend

The fluorescence lifetime of lipofuscin granule fluorophores contained in the retinal pigment epithelium cells from human cadaver eyes in normal state and in the case of visualized pathology

A comparative analysis of fluorescence lifetime of lipofuscin granule fluorophores contained in the retinal pigment epithelium cells from human cadaver eyes in normal state and in the case of visualized pathology was carried out. Measurements of fluorescence lifetimes of bis-retinoids and their photooxidation and photodegradation products were carried out using the method of counting time-correlated photons. Comparative analysis showed that, in the case of visualized pathology, the contribution of photooxidation and photodegradation products of bis-retinoids to the total fluorescence of the retinal pigment epithelium cell suspension increases in comparison with the norm.

Time-of-flight secondary ion mass spectrometry to assess spatial distribution of A2E and its oxidized forms within lipofuscin granules isolated from human retinal pigment epithelium

Lipofuscin granules accumulate in the cells of retinal pigment epithelium with age, particularly in patients with hereditary diseases. These granules are heterogeneous, being composed of mixtures of proteins and lipids, including more than 21 different fluorescent compounds. Bisretinoids and their photo-oxidation and photodegradation products represent the main source of lipofuscin fluorescence and exhibit phototoxic properties. This study used time-of-flight secondary ion mass spectrometry (ToF–SIMS) with in-depth probing to assess the depth distribution of N-retinylidene-N-retinylethanolamine (A2E) and its singly and doubly oxidized forms (A2E-ox and A2E-2ox, respectively) within lipofuscin granules and in their surface layer (lipid membrane). ToF–SIMS showed that A2E and its oxidized forms were uniformly distributed throughout lipofuscin granules but were not present at the membrane surface layer. This finding is important for understanding the process involved in the formation of lipofuscin granules and in their toxicity.