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Microsecond Time-Resolved Circular Dichroism of Rhodopsin Photointermediates

机译：视紫红质光中间体的微秒时间分辨圆二色性

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摘要

Time-resolved circular dichroism measurements, over a spectral range from 300 to 700 nm, werenmade at delays of 5, 100, and 500 μs after room-temperature photoexcitation of bovine rhodopsin in a laurylnmaltoside suspension. The purpose was to provide more structural information about intermediate states innthe activation of rhodopsin and other G protein-coupled receptors. In particular, information was soughtnabout photointermediates that are isochromic or nearly isochromic in their unpolarized absorbance. Thencircular dichroism spectrum of lumirhodopsin, obtained after correcting the 5 μs difference CD data for thenbleached rhodopsin, was in reasonable agreement with the lumirhodopsin CD spectrum obtained previouslynby thermal trapping at -76 u0001C. Similarly, the metarhodopsin II spectrum obtained with a 500 μs delay wasnalso in agreement with the results of previous work on the temperature-trapped form of metarhodopsin II.nHowever, the CD of the mixture formed with a 100 μs delay after photoexcitation, whose only visiblenabsorbing component is lumirhodopsin, could not be accounted for near 480 nm in terms of the initiallynformed, 5 μs lumirhodopsinCDspectrum. Thus, theCDspectrum of lumirhodopsin changes on the time scalenfrom 5 to 100 μs, showing reduced rotational strength in its visible band, possibly associated with either anprocess responsible for a small spectral shift that occurs in the lumirhodopsin absorbance spectrum at earlierntimes or the Schiff base deprotonation-reprotonation which occurs during equilibration of lumirhodopsinnwith the Meta I380 photointermediate. Either explanation suggests a chromophore conformation changenclosely associated with deprotonation which could be the earliest direct trigger of activation.
机译：在室温下将牛视紫红质在月桂苷悬浮液中进行室温光激发后，在300至700 nm的光谱范围内，在5、100和500μs的延迟下进行时间分辨的圆二色性测量。目的是提供关于视紫红质和其他G蛋白偶联受体活化中间状态的更多结构信息。特别地，寻求关于其非极化吸收为等色或接近等色的光中间体的信息。然后，校正了随后漂白的视紫红质的5μs差异CD数据后获得的鲁美视紫红质的圆二色性光谱与先前通过-76 u0001C热阱获得的鲁美视紫红质CD光谱合理地一致。类似地，延迟500μs获得的金属紫红质II光谱也与先前关于温度捕获形式的甲基视紫红质II的研究结果相符。然而，混合物的CD在光激发后形成了100μs的延迟，其唯一可见光吸收成分是发光素视紫红质，就最初形成的5μs发光素视紫红质CD光谱而言，不能解释为接近480 nm。因此，发光素视紫红质的CD光谱在5到100μs的时间尺度上变化，显示出其可见光带的旋转强度降低，这可能与导致发光素视紫红质吸收光谱在较早的时间内发生小的光谱偏移的过程或席夫碱去质子化有关。在用Meta I380光中间体平衡发光发光蛋白的过程中发生的质子化。两种解释都表明与去质子密切相关的生色团构象可能是最早的激活直接触发。

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