Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, Missouri.
School of Molecular Sciences and The Biodesign Institute, Arizona State University, Tempe, Arizona.
Biophys J. 2019 Jul 9;117(1):66-73. doi: 10.1016/j.bpj.2019.05.026. Epub 2019 Jun 7.
The cyanine dye Cy3 is a popular fluorophore used to probe the binding of proteins to nucleic acids as well as their conformational transitions. Nucleic acids labeled only with Cy3 can often be used to monitor interactions with unlabeled proteins because of an enhancement of Cy3 fluorescence intensity that results when the protein contacts Cy3, a property sometimes referred to as protein-induced fluorescence enhancement (PIFE). Although Cy3 fluorescence is enhanced upon contacting most proteins, we show here in studies of human replication protein A and Escherichia coli single-stranded DNA binding protein that the magnitude of the Cy3 enhancement is dependent on both the protein as well as the orientation of the protein with respect to the Cy3 label on the DNA. This difference in PIFE is due entirely to differences in the final protein-DNA complex. We also show that the origin of PIFE is the longer fluorescence lifetime induced by the local protein environment. These results indicate that PIFE is not a through space distance-dependent phenomenon but requires a direct interaction of Cy3 with the protein, and the magnitude of the effect is influenced by the region of the protein contacting Cy3. Hence, use of the Cy3 PIFE effect for quantitative studies may require careful calibration.
菁染料 Cy3 是一种常用的荧光团，用于探测蛋白质与核酸的结合以及它们的构象转变。仅用 Cy3 标记的核酸通常可用于监测与未标记蛋白质的相互作用，因为当蛋白质与 Cy3 接触时，Cy3 荧光强度会增强，这种性质有时被称为蛋白质诱导的荧光增强 (PIFE)。尽管 Cy3 荧光在与大多数蛋白质接触时都会增强，但我们在对人复制蛋白 A 和大肠杆菌单链 DNA 结合蛋白的研究中表明，Cy3 增强的幅度既取决于蛋白质本身，也取决于蛋白质相对于 DNA 上 Cy3 标记的方向。这种 PIFE 的差异完全归因于蛋白质-DNA 复合物的最终结构的差异。我们还表明，PIFE 的起源是由局部蛋白质环境诱导的更长的荧光寿命。这些结果表明，PIFE 不是一个依赖于空间距离的现象，而是需要 Cy3 与蛋白质的直接相互作用，并且该效应的幅度受与 Cy3 接触的蛋白质区域的影响。因此，使用 Cy3 PIFE 效应进行定量研究可能需要仔细校准。
