Single-Molecule Fluorescence Spectroscopy of 4-Cyanotryptophan and Its Application in Photoinduced Electron Transfer Fluorescence Correlation Spectroscopy

Single-molecule fluorescence spectroscopy is a powerful tool to study the conformational dynamics of proteins. However, the protein in question was normally labeled with one or two fluorescent dyes, which may induce undesirable perturbation toward the protein's native property. Therefore, a long-sought goal is to use tryptophan, a natural fluorescent amino acid, for single-molecule fluorescence studies. Nevertheless, this has been shown to be challenging. Herein, we demonstrate that 4-cyanotryptophan, a genetically incorporable blue fluorescent amino acid with a large fluorescence quantum yield (>0.8), is bright enough for single-molecule fluorescence detection. In particular, we demonstrate its utility in florescence correlation spectroscopy (FCS) and use it to probe the conformational dynamics of the Pin1 WW domain via PET-FCS. We find that the transient hydrophobic interactions in the unfolded state of this protein occur on a time scale of ca. 10 μs, which provides insight into the earliest events in protein folding.