Trace fluorescent labeling for protein crystallization.
Trace fluorescent labeling for protein crystallization.
复制标题
DOI：
10.1107/s2053230x15008626
复制
发表时间：
2015-07
期刊：
影响因子：
--
通讯作者：
Ng J
中科院分区：
文献类型：
--
作者：
Pusey M;Barcena J;Morris M;Singhal A;Yuan Q;Ng J
在<0.5%的浓度下存在共价结合荧光探针不影响大分子结晶筛选实验的结果。此外，荧光可用于确定新的，不立即明显的铅结晶条件。荧光是帮助蛋白质结晶的有力工具。在痕量标记方法中，蛋白质用高量子产率可见波长荧光探针共价衍生。最终探针浓度通常标记≤0.20%的蛋白质分子，这已被证明不影响晶体成核或衍射质量。然后用通常的方法将标记的蛋白质用于平板筛选实验。由于蛋白质最密集的排列状态是晶体形式，因此晶体在荧光照射下显示为孔中最亮的物体。研究了微量荧光标记与未标记蛋白对筛选结果的影响，发现考虑到晶体成核过程的随机性，探针的存在并不影响所得结果。使用荧光可以实现其他效果。即使埋在沉淀物中，晶体也清晰可见。这种方法还发现了“隐藏”的引线，以亮点的形式，在单次优化试验中，发现的约30%的引线被优化为晶体。使用可见荧光还可以选择绕过干扰物质的颜色，并且筛选材料不必是紫外线透明的。
The presence of a covalently bound fluorescent probe at a concentration of <0.5% does not affect the outcome of macromolecule crystallization screening experiments. Additionally, the fluorescence can be used to determine new, not immediately apparent, lead crystallization conditions. Fluorescence can be a powerful tool to aid in the crystallization of proteins. In the trace-labeling approach, the protein is covalently derivatized with a high-quantum-yield visible-wavelength fluorescent probe. The final probe concentration typically labels ≤0.20% of the protein molecules, which has been shown to not affect the crystal nucleation or diffraction quality. The labeled protein is then used in a plate-screening experiment in the usual manner. As the most densely packed state of the protein is the crystalline form, then crystals show as the brightest objects in the well under fluorescent illumination. A study has been carried out on the effects of trace fluorescent labeling on the screening results obtained compared with nonlabeled protein, and it was found that considering the stochastic nature of the crystal nucleation process the presence of the probe did not affect the outcomes obtained. Other effects are realised when using fluorescence. Crystals are clearly seen even when buried in precipitate. This approach also finds ‘hidden’ leads, in the form of bright spots, with ∼30% of the leads found being optimized to crystals in a single-pass optimization trial. The use of visible fluorescence also enables the selection of colors that bypass interfering substances, and the screening materials do not have to be UV-transparent.