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首页> 外文期刊>Biochemistry >Rhodopsin in Nanodiscs Has Native Membrane-like Photointermediates
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Rhodopsin in Nanodiscs Has Native Membrane-like Photointermediates

机译：纳米圆盘中的视紫红质具有天然的膜状光中间体

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Time-dependent studies of membrane protein function arenhindered by extensive light scattering that impedes application of fastnoptical absorbance methods. Detergent solubilization reduces light scat-ntering but strongly perturbs rhodopsin activation kinetics. Nanodiscs maynbe a better alternative if they can be shown to be free from the seriousnkinetic perturbations associated with detergent solubilization. To resolventhis, wemonitored absorbance changes due to photointermediates formednon the microsecond to hundred millisecond time scale after excitation ofnbovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes asnwell as to those that form in lauryl maltoside suspensions at 15 and 30 u0001C over a pH range from 6.5 to 8.7. Time-resolved differencenspectra were collected from 300 to 700 nm at a series of time delays after photoexcitation and globally fit to a sum of time-decayingnexponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. Atnthe temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similarnto those that form in nativemembrane, in particular displaying the normal forward shift of theMeta I480 aMeta II equilibriumwithnincreased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergentnsuspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments withnrhodopsinmutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterizednin nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.
机译：膜蛋白功能的时间依赖性研究受到广泛的光散射的阻碍，这阻碍了快速正谱吸收法的应用。洗涤剂的增溶作用减少了光的散射，但强烈干扰了视紫红质的活化动力学。如果可以证明纳米光盘没有与洗涤剂增溶相关的严重的动力学扰动，则它们可能不是更好的选择。为了解决这个问题，我们监测了由于吸收牛视紫红质纳米圆盘后几微秒至几百毫秒时间范围内形成的光中间物而引起的吸光度变化，并将它们与低渗洗涤天然膜中形成的光中间物以及在15和30 u0001C的月桂基麦芽糖苷混悬液中形成的光中间物进行了比较。 pH范围为6.5至8.7。在光激发后的一系列时间延迟下，从300至700 nm处收集到时间分辨的差光谱，并全局拟合为时间衰减指数项的总和，并根据指数项的光谱系数确定存在的光中间体。在所研究的温度和pH值下，视紫红质在纳米圆盘中进行光激发后形成的光中间体与在天然膜中形成的光中间体极为相似，特别是在温度升高和pH降低的情况下，显示了天然I480 aMeta II平衡的正常前移，这发生在天然膜中，但未观察到在月桂基麦芽糖苷去污剂悬浮液中。这些结果是使用视紫红质突变体进行光学实验所需的纳米圆盘中视紫红质的量获得的。这项工作表明，可以在纳米圆盘上表征晚期的，重要的生理上的视紫红质光中间体，该圆盘提供了去污剂的优异光学性能，而不会干扰激活序列。

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  • 来源
    《Biochemistry》 |2011年第22期|p.5086-5091|共6页
  • 作者

    Hisao Tsukamoto Istvan Szundi James W. Lewis David L. Farrens and David S. Kliger;

  • 作者单位

    †Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, Oregon 97239, United States‡Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, United States;

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