Rhodopsin in nanodiscs has native membrane-like photointermediates.
Rhodopsin in nanodiscs has native membrane-like photointermediates.
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纳米圆盘中的视紫红质具有天然的膜状光中间体。
DOI：
10.1021/bi200391a
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发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Kliger,DavidS
中科院分区：
文献类型：
--
作者：
Tsukamoto,Hisao;Szundi,Istvan;Lewis,JamesW;Farrens,DavidL;Kliger,DavidS
膜蛋白功能的时间依赖性研究受到广泛的光散射的阻碍，这阻碍了快速光学吸收方法的应用。洗涤剂增溶减少光散射，但强烈干扰视紫红质活化动力学。如果纳米盘可以被证明不受与去污剂溶解相关的严重动力学扰动，则它们可能是更好的替代品。为了解决这个问题，我们监测了由于在牛视紫红质纳米盘激发后在微秒至百毫秒的时间尺度上形成的光中间体引起的吸光度变化，并将它们与在低渗洗涤的天然膜中形成的光中间体以及在15和30 °C下在6.5至8.7的pH范围内在月桂基麦芽糖苷悬浮液中形成的光中间体进行比较。从300至700 nm的时间分辨的差光谱收集在一系列的时间延迟后，光激发和全局拟合的时间衰减指数项的总和，和存在的光中间体的指数项的光谱系数确定。在所研究的温度和pH下，在纳米盘中的视紫红质的光激发之后形成的光中间体与在天然膜中形成的光中间体极其相似，特别是显示出Meta I480-Meta II平衡随温度升高和pH降低的正常前向位移，这发生在天然膜中，但在月桂基麦芽糖苷洗涤剂悬浮液中未观察到。这些结果是使用纳米盘中视紫红质的量获得的，这是使用视紫红质突变体进行光学实验所需的。这项工作表明，后期，生理上重要的视紫红质光中间体可以在nanodisks，它提供了洗涤剂的上级光学性能，而不扰乱激活序列的特点。
Time-dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization. To resolve this, we monitored absorbance changes due to photointermediates formed on the microsecond to hundred millisecond time scale after excitation of bovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes as well as to those that form in lauryl maltoside suspensions at 15 and 30 °C over a pH range from 6.5 to 8.7. Time-resolved difference spectra were collected from 300 to 700 nm at a series of time delays after photoexcitation and globally fit to a sum of time-decaying exponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. At the temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similar to those that form in native membrane, in particular displaying the normal forward shift of the Meta I480⇄ Meta II equilibrium with increased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergent suspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments with rhodopsin mutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterized in nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.