To expand the spectroscopic utility of the non-natural amino acid p-cyanophenylalanine (Phe(CN)), we examine the quenching efficiencies of a series of commonly encountered anions toward its fluorescence. We find that iodide exhibits an unusually large Stern-Volmer quenching constant, making it a convenient choice in Phe(CN) fluorescence quenching studies. Indeed, using the villin headpiece subdomain as a test-bed we demonstrate that iodide quenching of Phe(CN) fluorescence offers a convenient means to reveal protein conformational heterogeneity. Furthermore, we show that the amino group of PheCN strongly quenches its fluorescence, suggesting that Phe(CN) could be used as a local pH sensor. (C) 2013 Elsevier B. V. All rights reserved.