Halide and proton binding kinetics of yellow fluorescent protein variants

A T203Y substitution in green fluorescent protein causes a red shift in emission to yield a class of mutants known as yellow fluorescent protein (YFP). Many of these YFP mutants bind halides with affinities in the millimolar range, which often results in the chromophore pK values being shifted into the physiological range. While such sensitivities may be exploited for halide and pH sensors, it is desirable to reduce such environmental sensitivities in other studies, such as in Förster resonance energy transfer probes to measure conformational changes within fusion proteins. Venus and Citrine are two such variants that have been developed with much reduced halide sensitivities. Here we compare the kinetics of halide binding, and the coupled protonation reaction, for several YFP variants and detect slow kinetics (dissociation rate constants in the range of 0.1-1 s(-1)), indicative of binding to an internal site, in all cases. The effective halide affinity for Venus and Citrine is much reduced compared with that of the original YFP 10C construct, primarily through a reduced association rate constant. Nuclear magnetic resonance studies of YFP 10C confirm halide binding occurs on a slow time scale (<4 s(-1)) and that perturbations in the chemical shift occur throughout the sequence and structure.