Rhodopsin Photointermediates in Two-Dimensional Crystals at Physiological Temperatures
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- 作者：Istvan Szundi ; Jonathan J. Ruprecht ; Jacqueline Epps ; Claudio Villa ; Trevor E. Swartz ; James W. Lewis ; Gebhard F. X. Schertler ; and David S. Kliger
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：April 18, 2006
- 年：2006
- 卷：45
- 期：15
- 页码：4974 - 4982
- 全文大小：208K
- 年卷期：v.45,no.15(April 18, 2006)
- ISSN：1520-4995
文摘
Bovine rhodopsin photointermediates formed in two-dimensional (2D) rhodopsin crystalsuspensions were studied by measuring the time-dependent absorbance changes produced after excitationwith 7 ns laser pulses at 15, 25, and 35 
C. The crystalline environment favored the Meta I480photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membranesuspensions at 35 C and its decay to a 380 nm absorbing species being less complete than it is in thenative membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the380 nm absorbing product of Meta I480 decay did not display the anomalous pH dependence characteristicof classical Meta II in the native disk membrane. Crystal suspensions bleached at 35 C and quenched to19 C showed that a rapid equilibrium existed on the ~1 s time scale, which suggests that the unprotonatedpredecessor of Meta II in the native membrane environment (sometimes called MIIa) forms in 2D rhodopsincrystals but that the non-Schiff base proton uptake completing classical Meta II formation is blockedthere. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and doesnot result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data ofthe 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changesand the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in thenative membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach dueto the random orientation of linearly dichroic crystals relative to the linearly polarized laser was exploredas a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nmproduct of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activationsuggests that determination of its structure would be of interest.
C. The crystalline environment favored the Meta I480photointermediate, with its formation from Lumi beginning faster than it does in rhodopsin membranesuspensions at 35 C and its decay to a 380 nm absorbing species being less complete than it is in thenative membrane at all temperatures. Measurements performed at pH 5.5 in 2D crystals showed that the380 nm absorbing product of Meta I480 decay did not display the anomalous pH dependence characteristicof classical Meta II in the native disk membrane. Crystal suspensions bleached at 35 C and quenched to19 C showed that a rapid equilibrium existed on the ~1 s time scale, which suggests that the unprotonatedpredecessor of Meta II in the native membrane environment (sometimes called MIIa) forms in 2D rhodopsincrystals but that the non-Schiff base proton uptake completing classical Meta II formation is blockedthere. Thus, the 380 nm absorbance arises from an on-pathway intermediate in GPCR activation and doesnot result from early Schiff base hydrolysis. Kinetic modeling of the time-resolved absorbance data ofthe 2D crystals was generally consistent with such a mechanism, but details of kinetic spectral changesand the fact that the residuals of exponential fits were not as good as are obtained for rhodopsin in thenative membrane suggested the photoexcited samples were heterogeneous. Variable fractional bleach dueto the random orientation of linearly dichroic crystals relative to the linearly polarized laser was exploredas a cause of heterogeneity but was found unlikely to fully account for it. The fact that the 380 nmproduct of photoexcitation of rhodopsin 2D crystals is on the physiological pathway of receptor activationsuggests that determination of its structure would be of interest.