Fluorescence Dynamics of N-Terminal Tryptofan-X Residues in Polypeptide: pH Response

The influence of the second amino acid on the pH response of the genetically encoded pH indicators is studied. The second Trp has been substituted by Ala and Glu to reduce self-quenching. Compared with the previously reported Trp-Trp-Ala-Ser (WWAS), the two new tetrapeptides, Trp-Ala-Ala-Ser (WAAS) and Trp-Glu-Ala-Ser (WEAS), have higher fluorescence quantum yields, longer fluorescence lifetimes, and more sensitive pH responses. As novel genetically encoded pH indicators, the N-terminal Trp-Ala and Trp-Glu could be fused to proteins for monitoring the environmental pH values during the studies of functional proteins.