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 (Phe_CN) 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 Phe_CN, and the resultant FRET pairs offer certain advantages over Phe_CN-Trp. For example, the fluorescence spectrum of 7AW is sufficiently separated from that of Phe_CN, making it straightforward to decompose the FRET spectrum into donor and acceptor contributions. Moreover, we show that Phe_CN, 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.