Abstract:
:The green fluorescent protein YFP-H148Q is sensitive to halides by a mechanism 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(-). Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high-throughput screening, and for measurement 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 clones revealed improved anion sensitivities with K(d) down to 2 mM for I(-) (I152L), 40 mM for Cl(-) (V163S), and 10 mM for NO(3)(-) (I152L). The anion-sensitive mechanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transmembrane conductance regulator chloride channel.
journal_name
FEBS Lettjournal_title
FEBS lettersauthors
Galietta LJ,Haggie PM,Verkman ASdoi
10.1016/s0014-5793(01)02561-3keywords:
subject
Has Abstractpub_date
2001-06-22 00:00:00pages
220-4issue
3eissn
0014-5793issn
1873-3468pii
S0014-5793(01)02561-3journal_volume
499pub_type
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