• 생명과학회지
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• 제17권6호통권86호
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• pp.783-790
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• 2007

G protein-coupled receptor, (GPCR)는 세포외부의 신호를 인식 시 G 단백질을 활성화시켜 신호를 전달하며 kinase에 의한 인산화를 통하여 지속적인 신호전달을 억제한다. 외부 신호물질이 없는 조건에서도 활성을 나타내는 항활성 돌연변이종(CAM)은 GPCR의 신호전달 이상에 기인한 질병 치료나 활성화 구조변화의 좋은 연구대상이다. 희미한 빛을 인식하는 시각수용체인 로돕신의 CAM으로는 salt bridge에 직접적인 영향을 미치는 돌연변이인 G90D, El13Q, 그리고 K296E와, 직접적인 영향이 없는 돌연변이인 E134q와 M25Y등 두 가지 계통의 종류가 알려져 있다. 본 연구에서는 각각의 돌연변이가 복합된 mutant를 구성하여 agonist와 inverse agonist에 대한친화도와 로돕신 kinase에 대한 활성을 조사하여 각 종에서의 구조변화의 차이를 분석하였다. 로돕신 mutant의constitutive activity는 all-trans-retinal에 대한 친화도에 비례하며 11-cis-retinal에 대한 친화도와는 역상관 관계를 보여준다. 같은 계통에 속하는 돌연변이가 합쳐진 복합 mutant는 단일 mutant에 비하여 미약한 정도의 로돕신 kinase 항활성화 증가를 보여주나, 다른 계통에 속하는 두 가지 돌연변이가 합쳐진 mutant는 항활성화가 크게 증가되었음을 보여주었다. 이 결과는 다른 계통에 속하는 mutant에서는 상이한 구조변화가 일어나며 로돕신이완전한 활성화에 이르기 위해서는 최소한 두 가지 종류의 돌연변이에 의하여 생기는 구조변화들이 함께 일어나야함을 의 미 한다. G protein 활성화와 유사한 항활성화 분석 결과는 rhodopsin kinase가 인식하는 로돕신의 활성화상태 구조가 G protein이 인식하는 구조와 유사함을 의미한다. 특히 가장 강한 활성을 나타내는 El13Q/E134Q/M257Y는 활성화상태 GPCR 단백질의 결정 시도에 이용 될 수 있을 것이다.

• BMB Reports
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• 제37권2호
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• pp.260-267
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• 2004

Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.

• Journal of Photoscience
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• 제2권1호
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• pp.19-25
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• 1995

Picosecond time-resolved and static protein fluorescence spectra and absorption spectra of octopus rhodopsin, a photorecepting protein, are measured and compared with those of bacteriorhodopsin, a photon-induced proton pumping protein, to understand the protein conformations and functions of octopus rhodopsin and its deprotonated photocycle intermediate. The bluer and weaker absorption of retinal indicates that octopus rhodopsin is better in thermal noise suppression but less efficient in light harvesting than bacteriorhodopsin. The protein fluorescence of octopus rhodopsin shows the characteristic of Trp only and the uantum efficiency and lifetime variations may result primarily from variations in the coupling strength with the retinal. The stronger intensity by four times and larger red shift by 12 nm of fluorescence suggest that octopus rhodopsin has more open and looser structure compared with bacteriorhodopsin. Fluorescence decay profiles reveal two decay components of 300 ps (60%) and 2 ns (40%). The deprotonation of protonated Schiff's base increases the shorter decay time to 500 ps and enhances the fluorescence intensity by 20%. The fluorescence and its decay time from Trp residues near retinal are influenced more by the deprotonation. The increase of fluorescence intimates that protein structure becomes loosened and relaxed further by the deprotonation of protonated Schiff's base. The driving force of sequential changes initiated by absorption of a photon is too exhausted after the deprotonation to return the intermediate to the ground state of the begun rhodopsin form.

• Journal of Photoscience
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• 제9권2호
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• pp.51-54
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• 2002

We propose a novel mechanism (Twist Sharing Mechanism) for the cis-trans photoisomerization of rhodopsin, based on the molecular dynamics (MD) simulation study. New things devised in our simulations are (1) the adoption of Mt. Fuji potentials in the excited state for twisting of the three bonds C9=C10, C11=C12 and C13=14 which are modeled using the detailed ab initio quantum chemical calculations and (2) to use the rhodopsin structure which was resolved recently by the X-ray crystallographic study. As a result, we found the followings: Due to the intramolecular steric hindrance between 20-methyl and 10-H in the retinal chromophore, the C12-C13 and C10-C11 bonds are considerably twisted counterclockwise in rhodopsin, allowing only counterclockwise rotation of the C11 =C12 in the excited state. The movement of 19-methyl in rhodopsin is blocked by the surrounding three amino acids, Thr 118, Met 207 and Tyr 268, prohibiting the rotation of C9=C10. As a result only all-trans form of the chromophore is obtainable as a photoproduct. At the 90$^{\circ}$ twisting of C11=C12 in the course of photoisomerization, twisting energies of the other bonds amount to about 20 kcal/mol. If the transition state for the thermal isomerization is assumed to be similar to this structure, the activation energy for the thermal isomerization around C11=C12'in rhodopsin is elevated by about 20 kcal/mol and the thermal isomerization rate is decelerated by 10$\^$-14/ times than that of the retinal chromophore in solution, protecting photosignal from the thermal noise.

• Journal of Photoscience
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• 제9권2호
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• pp.33-36
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• 2002

In vertebrate rhodopsins the glutamic acid at position 113 serves as a counterion to stabilize the protonated retinylidene Schiff base linkage and to shift the spectrum to the visible region. Invertebrate rhodopsins and retinochrome have the amino acid residue different from glutamic acid or asparatic acid at this position and therefore, these pigments may have a counterion at different position. We first investigated the counterion in retinochrome by site specific mutagenesis. The results showed that the counterion is the glutamic acid at position 181, where almost of all the pigments including vertebrate and invertebrate rhodopsins in the rhodopsin family have a glutamic acid or an aspartic acid. In vertebrate rhodopsins, however, Glu 181 does not act as a counterion, and the red-sensitive cone pigments have a histidine at this position, which serves as a chloride-binding site for red-shift of the absorption spectrum. These findings suggested that the role of Glu181 as a counterion may be weakened by the newly acquired counterion at position 113. Taken together with our recent studies on an invertebrate-type rhodopsin, the rhodopsin diversity was discussed from viewpoint of counterion.

• 한국전기전자재료학회논문지
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• 제20권1호
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• pp.80-85
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• 2007

We report the electro-optical properties of the sensory rhodopsin II using a near-field scanning microwave microscope(NSMM). Rhodopsin was known as a photoreceptor pigment with a retinal as a chromophore via a protonated Schiff base and consists of seven ${\alpha}-helical$ transmembrane segments. The sensory rhodopsin II, expressing E. coli UT5600 with endogenous retinal biosynthesis system and purified with $Ni^{-2}-NTA$ affinity chromatography in the presence of 0.02 % DM (Dodecyl Maltoside) from Natronomonas pharaonis. We measured the absorption spectra and the transients difference of sensory rhodopsin II from Natronomonas pharaonis using a UV/VIS spectrophotometer with Nd-Yag Laser (532 nm). The absorption spectra of NpSR II showed a typical rhodopsin spectrum with a left shoulder region and the photointermediates spectra of NpSR II-ground state (${\lambda}max=498\;nm$), NpSR II-M state (${\lambda}max=390\;nm$), and NpSR II-O state (${\lambda}max=550\;nm$) during the photocycle. The observed photocycle reaction was confirmed by measuring the microwave reflection coefficient $S_{11}$ at an operating frequency of f=3.93-3.95 GHz and compared with the results of a photocycle of NpSR II.

• 대한화학회지
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• 제6권1호
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• pp.32-35
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• 1962

Six frogs (Rana nigromaculata 25∼40 gm) were adapted to light for 2 hours. Then 0.02 ml of 3% nicotinamide and 0.02 ml of 0.6% folic acid were injected into the vitreous body of the right eye-ball, and 0.02 ml normal saline solution into the vitreous body of the left eye-ball respectively. After dark adaptation for an hour-their heads were cut off under a dim red light (650 $m{\mu}$). The retinae were removed from the left eye-ball for the control group and from the right for the test group respectively. Then rhodopsin was extracted from the retinae with 3 ml of 2% digitonin solution(pH = 7.0) for 17 hours at 0$^{\circ}C$ in the dark. The optical densities before and after the illumination of the extract were measured and compared with those of the control group. The results are as follows: 1) The group which had been injected with 0.02 ml of 3% nicotinamide solution had the promotive action on tile regeneration of rhodopsin in comparison with the control group. 2) The group which had been injected with 0.02 ml of 0.6% folic acid solution had the controlling action on the regeneration of rhodopsin in comparison with the control group.

• BMB Reports
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• 제41권7호
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• pp.555-560
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• 2008

The interaction of the rod GTP binding protein, Transducin ($G_t$), with bleached Rhodopsin ($R^*$) was investigated by measuring radiolabeled guanine nucleotide binding to and release from soluble and/or membrane-bound Gt by reconstituting $G_t$ containing bound GDP ($G_t$-GDP) or the hydrolysis-resistant GTP analog guanylyl imidodiphosphate ($G_t$-p[NH]ppG) with $R^*$ under physiological conditions. Release of GDP and p[NH]ppG from $G_t$ occurred to the same extent and with the same light sensitivity both in the presence and absence of added GTP. Significant amounts of $G_t$ without bound nucleotide ($G_{t^-}$) were generated. When ROS containing bleached rhodopsin ($R^*$) were centrifuged in low ionic strength buffer, $G_{t^-}$ remained associated with the membrane fraction, whereas $G_t$-GDP remained in the soluble fraction. These results suggest that $G_t$-GDP and $G_t$-p[NH]ppG have similar affinities for $R^*$. The results also suggest that $G_{t^-}$, rather than $G_t$-GDP, is the moiety which exhibits tight, "light-induced" binding to rhodopsin.

• Journal of Photoscience
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• 제9권2호
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• pp.1-4
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• 2002

Hula-twist is a volume-conserving photoisomerization reaction mechanism postulated in 1985 to account for the rapid photoisomerization of the retinyl chromophore in rhodopsin. The requisite stereochemical consequence of simultaneous isomerization of a double bond and an adjacent single bond has recently been demonstrated in isomerization of pre-vitamin D in an organic glass and by many other examples of organic systems already reported in the literature This paper reports the consequence in applying the mechanism to the primary photochemical process of several photosensitive biopigments: bilirubin, photoactive yellow protein, bacteriorhodopsin and rhodopsin. It is shown that the anchored nature of the chromophores must first be taken into consideration.

• Journal of Photoscience
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• 제10권1호
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• pp.81-87
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• 2003

Based on structural information provided by recently reported crystal structures of rhodopsin, we present rationales for the regiospecific protein perturbation on the previously reported $\^$19/F chemical shifts of the vinyl and trifluoromethylrhodopsins and their photoproducts. The crystal structures also suggest that H-bonding is a likely cause for the earlier reported regiospecific photoisomerization of the 10-fluororhodopsins. Photoisomerization was revealed by chemical shift of the photoproducts. Additionally, possible use of 3-bond F,F coupling constants for following photoisomerization of retinal-binding proteins is discussed.