Research Article

One key mutation introducing a second cysteine residue in place of asparagine (CreiLOV_N41C) significantly altered metal affinity and selectivity, yielding a zinc sensor. The fluorescence intensity and lifetime of CreiLOV_N41C were reversibly quenched by Zn2+ ions with a biologically relevant affinity (apparent dissociation constant, Kd, of 1 nM). Copper quenching of CreiLOV_N41C was retained but with several orders of magnitude higher affinity than CreiLOV (Kd = 0.066 fM for Cu2+, 5.4 fM for Cu+) and partial reversibility.