Non-natural amino acid fluorophores for one- and two-step fluorescence resonance energy transfer applications

Fluorescence resonance energy transfer (FRET) provides a powerful means to study protein conformational changes. However, the incorporation of an exogenous FRET pair into a protein could lead to undesirable structural perturbations of the native fold. One of the viable strategies to minimizing such perturbations is to use non-natural amino acid-based FRET pairs. Previously, we showed that p-cyanophenylalanine (PheCN) and tryptophan (Trp) constitute such a FRET pair, useful for monitoring protein folding–unfolding transitions. Here we further show that 7-azatryptophan (7AW) and 5-hydroxytryptophan (5HW) can also serve as a FRET acceptor to PheCN, and the resultant FRET pairs offer certain advantages over PheCN–Trp. For example, the fluorescence spectrum of 7AW is sufficiently separated from that of PheCN, making it straightforward to decompose the FRET spectrum into donor and acceptor contributions. Moreover, we show that PheCN, Trp, and 7AW can be used together to form a multi-FRET system, allowing more structural information to be extracted from a single FRET experiment. The applicability of these FRET systems is demonstrated in a series of studies where they are employed to monitor the urea-induced unfolding transitions of the villin headpiece subdomain (HP35), a designed ββα motif (BBA5), and the human Pin1 WW domain.