Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Nat Struct Mol Biol. 2019 Sep;26(9):802-807. doi: 10.1038/s41594-019-0274-2. Epub 2019 Sep 5.
Conformational changes within typical protein molecules are rapid and small, making their quantitative resolution challenging. These changes generally involve rotational motions and may thus be resolved by determining changes in the orientation of a fluorescent label that assumes a unique orientation in each conformation. Here, by analyzing fluorescence intensities collected using a polarization microscope at a rate of 50 frames per second, we follow the changes of 10-16° in the orientation of a single bifunctional rhodamine molecule attached to a regulator of conductance to K (RCK) domain of the MthK channel, and thus, the transitions between its three conformational states, with effective standard deviation (σ) of 2-5°. Based on available crystal structures, the position of the fluorophore's center differs by 3.4-8.1 Å among the states. Thus, the present approach allows the resolution of protein conformational changes involving ångström-scale displacements.
典型蛋白质分子内的构象变化迅速且微小，这使得对其进行定量解析具有挑战性。这些变化通常涉及旋转运动，因此可以通过确定荧光标记物在每个构象中呈现独特取向的变化来解决。在这里，我们通过以每秒 50 帧的速率使用偏光显微镜分析荧光强度，跟踪附着在 MthK 通道电导调节剂（RCK）域上的单个双功能罗丹明分子的取向变化 10-16°，从而跟踪其三个构象状态之间的转变，其有效标准偏差（σ）为 2-5°。基于现有的晶体结构，荧光团中心在不同状态下的位置差异为 3.4-8.1 Å。因此，本方法可以解析涉及埃尺度位移的蛋白质构象变化。
