Modulation of p-Cyanophenylalanine Fluorescence by Amino Acid Side Chains and Rational Design of Fluorescence Probes of α-Helix Formation

Fluorescence spectroscopy is widely used to study proteins and peptides, with the vast majority of studies making use of the naturally occurring fluorophores, Tyr and Trp (1, 2). However, there is considerable interest in the incorporation of novel fluorophores, because they can offer site specific probes and, in many cases, they can be chosen to allow selective excitation and detection (3–7). Recently p-cyanophenylalanine (FCN) fluorescence has been developed as a robust probe of protein folding, protein-peptide interactions, protein-membrane interactions, ligand binding and amyloid formation (Figure 1) (8–15). Figure 1 General sequence of the peptides studied. (A) Several variants have an amidated C-terminus and a free N-terminus. (B) An additional three peptides GGFCNAA, GGFCNHA and GGFCNKA were synthesized with an acetylated N-terminus. (C) The structure of the p ... The utility of p-cyanophenylalanine is due to three features. First, it can be incorporated into proteins recombinantly, using the so called 21st pair methodology, or by solid-phase peptide synthesis (11, 16). Secondly, it represents a relatively conservative substitution for the aromatic amino acids since it is much more similar in shape and size than many other fluorophores and the polarity of the cyano group is between that of a methylene and an amide group. This intermediate polarity allows FCN to be accepted in both a hydrophobic and hydrophilic environments in a protein. Third, the photophysical properties of FCN nicely complement existing fluorophores. It can be selectively excited in the presence of Trp and Tyr and it forms a useful resonance energy transfer (RET) probe with both Tyr and Trp (7, 14, 17, 18). However, the factors that control its quantum yield are not completely understood. It is known that the fluorescence is high when the cyano group is hydrogen bonded and fluorescence can be quenched by RET to Tyr or Trp, but unfortunately the effect of other amino acid side-chains is not known (14, 19). A detailed understanding of the factors that control FCN fluorescence is required to fully exploit this very promising probe and to avoid misinterpretation. A striking example is provided by our recent application of FCN to study the folding of NTL9, a small globular protein. The fluorescence of FCN in NTL9 was very low in the folded state suggesting that the cyano group was sequestered from solvent, however IR measurements showed that it was exposed, and further investigation revealed that the FCN fluorescence was quenched by interactions with a Tyr side-chain in the native state and showed that the cyano group was, in fact, exposed to solvent (14). Here we systematically examine the ability of other amino acids and the termini of polypeptides to modulate FCN fluorescence by examining the fluorescence of FCN in a set of peptides of general sequence GGFCNXA where X represents Ala, Cys, His, Lys, Met, Asn, Arg, and Tyr. Based on these results we demonstrate how such interactions can be used as a highly sensitive probe of helix formation. We also show that free imidazole and hydroxide ion are effective quenchers of FCN fluorescence.