The metal dependent quenching of iq-FPs are similar to other tmFRET-based fluorescence systems. Like those systems, other quenching mechanisms including static quenching and electron transfer are possible. However, because static quenching requires physical contact of the metal and the fluorophore, and electron transfer usually occurs at distances, we believe these effects to be unlikely because no metals were observed inside the beta-barrel near the chromophore. Furthermore, our data exhibits the typical two components of quenching seen in tmFRET experiments and the close match between our data and the FRET-based models strongly suggests that FRET is the dominant quenching mechanism. The first component likely results from energy transfer between the metal bound to the engineered site and the second component is due to non-specific solution-phase quenching. This second component is observed even in FPs. From our FRETspecific signal we can estimate the distance between the metals and the chromophore. To our knowledge, this is the first direct distance measurement of an intact energy transfer system by both crystallography and fluorescence. For example, the probe could be used as an alternative to pH-sensitive GFP as a reporter for exocytosis and endocytosis. Specifically, adding a solution of copper to a synaptic terminal or cell and measuring the fraction of fluorescence quenched by copper would reveal the amount of iqFP-tagged membrane proteins released into the plasma membrane during exocytosis. Likewise, two-color imaging could be done on different proteins tagged with the same color probe. The individual signals could be isolated by taking advantage of their drastically different intensities in metal solutions. In a similar way, iq-FPs could be used to locate specific weak signals from a highly fluorescent non-specific background. We demonstrate that metal-ion-induced fluorescence changes of iq-FPs could be used as genetically encoded sensors. Metal concentrations are regulated and play important roles in biological systems. Accumulation of metal ions can cause misfolding or aggregation of proteins that are linked to neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. Thus, measuring the locations and concentrations of these ions is critical. Generally, the E. faecalis species challenges the boundary between commensal and pathogen: while several genetic traits that contribute to the virulence of E. faecalis have been characterized, none has appeared to be indispensable for its pathogenicity. A distinct trait in E. faecalis physiology, compared to other intestinal lactic acid bacteria, is its ability to persist and thrive in harsh environments, that include heat, acid, oxidative and hyperosmotic stress. It is thus conceivable that the intrinsic robustness of E. faecalis is significant to the pathogenic potential of this bacterium.