Tracking multiple conformations occurring on angstrom-and-millisecond scales in single amino-acid-transporter molecules.
Tracking multiple conformations occurring on angstrom-and-millisecond scales in single amino-acid-transporter molecules.
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作者：
Zhou Y;Lewis JH;Lu Z
大多数膜蛋白分子在从一种功能状态过渡到另一种功能状态时会发生构象变化。要了解这些变化的机制，需要有能力解决个别构象状态，其变化往往发生在毫秒和埃尺度。跟踪这些变化和获取足够大量的数据仍然具有挑战性。在这里，我们使用的氨基酸转运蛋白AdiC作为一个例子，以证明高分辨率的荧光偏振显微镜方法在跟踪膜蛋白的多态构象变化的应用。我们已经成功地解决了四个构象的AdiC通过监测的发射偏振变化的荧光标记，并量化其概率的存在下，其底物精氨酸的一系列浓度。所获得的数据足以确定所有的平衡常数，充分建立四个状态之间的能量关系。精氨酸在四个单独的构象测定的KD值是统计学上可比的，以前报道的整体KD使用等温滴定量热法测定。这表明本偏振显微镜方法的强大的分辨能力将能够获得所需的定量信息，用于理解预期的复杂的构象机制的基础上的转运蛋白的功能，以及其他膜蛋白。
Most membrane protein molecules undergo conformational changes as they transition from one functional state to another one. An understanding of the mechanism underlying these changes requires the ability to resolve individual conformational states, whose changes often occur on millisecond and angstrom scales. Tracking such changes and acquiring a sufficiently large amount of data remain challenging. Here, we use the amino-acid transporter AdiC as an example to demonstrate the application of a high-resolution fluorescence-polarization-microscopy method in tracking multistate conformational changes of a membrane protein. We have successfully resolved four conformations of AdiC by monitoring the emission-polarization changes of a fluorophore label and quantified their probabilities in the presence of a series of concentrations of its substrate arginine. The acquired data are sufficient for determining all equilibrium constants that fully establish the energetic relations among the four states. The KD values determined for arginine in four individual conformations are statistically comparable to the previously reported overall KD determined using isothermal titration calorimetry. This demonstrated strong resolving power of the present polarization-microscopy method will enable an acquisition of the quantitative information required for understanding the expected complex conformational mechanism underlying the transporter’s function, as well as those of other membrane proteins.