Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
PLoS One. 2018 Jun 1;13(6):e0198416. doi: 10.1371/journal.pone.0198416. eCollection 2018.
The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.
乳糖操纵子阻遏蛋白 LacI 长期以来一直是细菌转录因子的典范。然而， LacI 快速定位其在细菌染色体上的同源结合位点的机制仍难以捉摸。单分子荧光成像方法非常适合研究这些机制，但依赖于 LacI 的功能兼容荧光标记。由于其体积小和有利的光物理特性，合成荧光团特别适合用于蛋白质荧光标记。合成荧光团通常使用马来酰亚胺化学将其连接到天然存在的半胱氨酸残基上。为了进行具有马来酰亚胺荧光团的特异性和功能兼容标记，目标蛋白通常需要重新设计，以去除不需要的天然半胱氨酸，并在更适合荧光团附着的位置引入半胱氨酸。然后可以进行生化筛选，以在染料标记前后探测重新设计的蛋白质的功能活性。在这里，我们报告了 LacI 的基于诱变的重新设计，以实现与马来酰亚胺荧光团的功能兼容标记。为了在 LacI 中提供一个易于接近的标记位点，我们在 LacI 的 DNA 结合头部引入了一个位于位置 28 的单个半胱氨酸残基，并将两个天然半胱氨酸替换为丙氨酸，因为在这些位置用大取代基衍生会损害蛋白质的活性。我们发现，重新设计的 LacI 在体外和体内都保持着强大的活性，只要位置 281 的第三个天然半胱氨酸保留在 LacI 中。在全内反射显微镜测定中，我们观察到单个 Cy3 标记的 LacI 分子与固定的 DNA 结合，该 DNA 含有同源 O1 操纵子序列，表明染料标记的 LacI 具有功能活性。因此，我们已经能够生成功能性荧光标记的 LacI，可用于在单分子水平上揭示 LacI 靶标搜索的机制细节。
