Green fluorescent protein-based halide indicators with improved chloride and iodide affinities

The green fluorescent protein YFP-H148Q is sensitive to halides by a mechan ism involving halide binding and a shift in pK(a), However, a limitation of YFP-H148Q is its low halide sensitivity, with K-d > 100 mM for Cl-. Indica tors with improved sensitivities are needed for cell transport studies, par ticularly in drug discovery by high-throughput screening, and for measureme nt of Cl- concentration in subcellular organelles, YFP-H148Q libraries were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated, An automated procedure was developed to screen bacterial colonies for improved halide sensitivity. Analysis of 1536 clone s revealed improved anion sensitivities with K-d down to 2 mM for I- (I152L ), 40 mM for Cl- (V163S), and 10 mM for NO3- (I152L), The anion-sensitive m echanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transm embrane conductance regulator chloride channel, (C) 2001 Federation of Euro pean Biochemical Societies. Published by Elsevier Science B.V. All rights r eserved.