• Bulletin of the Korean Chemical Society
• /
• 제25권11호
• /
• pp.1676-1680
• /
• 2004

We demonstrate that wide-angle X-ray scattering pattern from photoactive yellow protein (PYP) in solution using a high flux third generation synchrotron X-ray source reflects not only the overall structure, but also fine structures of the protein. X-ray scattering data from PYP in solution have been collected in q ranges from 0.02 ${\AA}^{-1}$ to 2.8 ${\AA}^{-1}$. These data are sensitive to the protein structure and consistent with the calculation based on known crystallographic atomic coordinates. Theoretical scattering patterns were also calculated for the intermediates during the photocycle of PYP to estimate the feasibility of time-resolved wide-angle X-ray scattering experiments on such proteins. These results demonstrate the possibility of using the wide-angle solution X-ray scattering as a quantitative monitor of photo-induced structural changes in PYP.

• 미생물학회지
• /
• 제47권1호
• /
• pp.22-29
• /
• 2011

Chromophore로 레티날(vitamin A)을 사용하는 막 단백질인 로돕신은 7개의 막 단백질로 이루어진다. 최근 광화학/생물리학 측정 방법이 다양해지면서, 그에 따른 새로운 특성도 함께 다양하게 보고되고 있다. 하지만, 다양한 측정 환경에 따른 그 광화학/생물리학 특성의 차이점에 대한 비교연구는 없는 상태이다. 첫째, 빛 에너지를 이용하여 수소 이온 펌프 역할 하는 것으로 잘 알려져 있는 roteorhodopsin (PR)은 해양 proteobacteria에서 발견 되었다. 둘째, 한 오페론에서 14 kDa 전달자와 같이 발현되면서 신호 전달 기능을 하는 Anabaena sensory rhodopsin (ASR)이 있다. 이에 본 연구에서는 이 두 미생물 로돕신을 이용하여 다양한 조건에서 그 차이를 살펴보았다. 각 단백질이 막에 끼어 있는 상태(membrane state), polyacrylamide gel에 고정되어 있는 상태, nonionic detergent일종인 DDM (n-dodecyl-${\beta}$-D-maltopyranoside)과, OG (octyl-${\beta}$-D-glucopyranoside)에 녹아 있는 상태, 좀더 자연상태와 유사한 환경을 위해 단백질만 깨끗하게 정제한 다음 다시 Escherichia coli의 지질인 DOPC (1,2-didecanoyl-sn-glycero-3-phosphocholine)에 재조립한 상태, 이렇게 5가지 조건에서 각각의 광화학/생물리학 특징을 흡수스펙트럼(absorption spectrum), 빛-어둠 차이 흡수스펙트럼(light-induced difference spectrum), 포토사이클(photocycle)을 측정하였다. 그 결과 DDM에 녹아 있는 PR과 ASR에서 각 다른 파장에서 M과 O state와 같은 선명한 photointermediate를 가지고, 가장 signal/noise 비율이 좋았다. 본 연구를 통해 막단백질의 다양한 측정 환경에 대한 그 특성의 차이점을 살펴봄으로써 앞으로 다양한 종류의 로돕신 연구에 기초 기반이 될 것으로 사료된다.

• Journal of Photoscience
• /
• 제9권2호
• /
• pp.317-319
• /
• 2002

All-optical switching has been demonstrated in bacteriorhodopsin (bR) based on nonlinear intensity induced excited state absorption. The transmission of a cw probe laser beam at 410 nm corresponding to the peak absorption of M state through a bR film is switched by a pulsed pump laser beam at 570 nm that corresponds to the maximum initial 8 state absorption. The switching characteristics have been analyzed using the rate equation approach considering all the six intermediate states (B, K, L, M, N and 0) in the bR photocycle. The switching characteristics are shown to be sensitive to life time of the M state, absorption cross-section of the 8 state at probe wavelength ($\sigma$ $\_$Bp/) and peak pump intensity. It has been shown that the probe laser beam can be completely switched off (100 % modulation) by the pump laser beam at relatively low pump powers, for $\sigma$$\_$Bp/ = O. The switching characteristics have been used to design all-optical NOT, OR, AND and the universal NOR and NAND logic gates for optical computing with two pulsed pump laser beams.

• Journal of Photoscience
• /
• 제9권2호
• /
• pp.114-117
• /
• 2002

Ultraviolet resonance Raman (UVRR) spectroscopy was used to elucidate the dynamic change of the protein structure of bacteriorhodopsin (BR) during the photocycle. The photointermediates minus light- adapted (LA) BR difference spectra show Trp difference signals, which are assigned to Trp189 or Trp182 on helix F by using the mutants, W182F and W189F. The Difference signals of Trp 182 indicates an increase in hydrogen bonding strength at the indole nitrogen and a large change in the side chain conformation (X$\^$2,1/ torsion angle) in the M$_1$ \longrightarrow M$_2$ transition. On the other hand, Trp189 shows an increased hydrophobic interaction. These results suggest that the tilt of helix F occurs in the M$_1$\longrightarrow M$_2$ transition. In the M$_2$ \longrightarrow N transition, the hydrophobic interaction of Trp182 decreases drastically, The decrease in hydrophobic interaction of Trp182 in the N state suggests an invasion of water molecules that promote the proton transfer from Asp96 to the Schiff base. Structural reorganization of the protein after the tilt of helix F may be important for efficient reprotonation of the Schiff base.

• Journal of Photoscience
• /
• 제9권2호
• /
• pp.314-316
• /
• 2002

In halobacterial membrane, pharaonis phoborhodopsin (or pharaonis sensory rhdopsin II, psRII) forms a complex with its transducer pHtrII. Flash-photolyis of D75N mutant did not yield M-intermediate but an O-like intermediate is observed. We examined the interaction between D75N of ppR and t-Htr (truncated pHtrII). These formed a complex in the presence of n-dodecyl-$\beta$-D-maltoside, and the association accelerated the decay of the 0 of D75N from 15 to 56 s$\^$-1/. From the decay time constants under varying ratios of D75N and t-Htr, n, the molar ratio of D75N/t-Htr in the complex, and K$\_$D/, the dissociation constant, were estimated. The value of n was unity and K$\_$D/ was estimated to 146 nM. This K$\_$D/ value can be considered as the association between the photo-intermediate and t-Htr, which is deduced by the method of estimation. Previously we (Photochem. Photobiol. 74, 489-494 (2001)) reported K$\_$D/ of 15 $\mu$M for the interaction between the wild-type and t-Htr by means of the change of M-decay rates. Therefore, this value should be the K$\_$D/ value for the interaction between M of the wild-type and t-Htr.

• Journal of Photoscience
• /
• 제9권2호
• /
• pp.311-313
• /
• 2002

X-ray structures of pharaonis phoborhodopsin (ppR) show the different direction of the side chain of Arg72 from that of the corresponding residue (Arg82) of bacteriorhodopsin, BR. For BR, this residue is considered to play an important role in the proton pumping. In order to investigate the role of Arg72 in ppR, we constructed Arg72 mutants of R72A, R72K and R72Q, and measured the photocycle and proton pumping activities. The pH-titration curves on the absorption maximum of the mutants were shifted to alkaline in comparison of that of the wild-type. This may imply the increase of pKa of D75, suggesting the presence of the (probably electric) interaction between D75 and Arg72. Rate constants of the M-decay were 3-7 times faster than that of the wild-type, and the time for the completion of the photocycling was also reduced. Using Sn0$_2$ electrode, the rate of transmembrane proton transport was measured upon illumination. The photo-induced proton pumping activities were estimated after the corrections that are the percentages of the associated form of D75 (which has no pumping activity) and the photocycling rates. R72A and R72Q showed the reduced activity while R72K did not reduce the activity.

• 생태와환경
• /
• 제41권spc호
• /
• pp.50-60
• /
• 2008

본 연구는 부영양 저수지에서 대발생하는 유해조류의 생물학적 제어를 위한 연구의 일환으로, 여과 섭식성 이매패 말조개(Unio crouglasiae)를 대상으로 수심 및 용존산소에 따른 여과율, 생존율 및 배설물 생산량을 조사하였다. 현장에서 말조개의 생존률은 20cm수심에서 가장 낮게 나타났고, 50cm와 80cm 수심에서 상대적으로 높게 나타났다. 말조개 생존율은 수온, pH, DO 농도와 높은 상관성을 나타냈고, 엽록소-${alpha}$ 농도, 부유물질 (SS)과 암모니아 농도와는 유의한 상관성을 나타내지 않았다. 수심실험에서 대조군의 엽록소-${alpha}$ 농도는 25%정도 감소한 반면, 수심 50, 80cm 처리군에서는 95% 정도까지 감소하였다. 말조개 여과율은 실험 18 hr까지 $0.15{\sim}0.20L\;gAFDW^{-1}hr^{-1}$ 범위를 보였으나, 이후 20 cm 처리군은 0.11 L $gAFDW^{-1}hr^{-1}$까지 감소한 반면, 50, 80cm 처리군에서는 각각 0.26, 0.30L $gAFDW^{-1}hr^{-1}$로 증가하였다. DO실험에서는 대조군의 엽록소-${alpha}$ 농도는 지속적으로 증가한 반면, 말조개를 투입한 모든 처리군에서는 감소하였다. 말조개 여과율은 8hr후 $0.5mgO_2\;L^{-1}$농도에서 0.46L $gAFDW^{-1}hr^{-1}$로 가장 높게 나타났으나 이후 DO농도에 따라 큰 차이를 나타내지 않았다. 말조개에 의한 배설물 생산은 실험 초기 DO농도가 낮을수록 높게 나타났지만, 시간이 지남에 따라 감소하여 실험 시작 24시간 후체는 3.0mg $gAFDW^{-1}hr^{-1}$ 정도로 비슷한 수준에 도달하였다. 이상의 결과를 종합할 때, 유해조류 제어를 위해 수층에서 말조개를 이용할 경우 용존산소에는 크게 영향을 받지 않으므로 표층보다는 좀 더 아래쪽(50, 80cm)에 설치하는 것이 효과적 일 것으로 사료된다.