Green fluorescent protein-based halide indicators with improved chloride and iodide affinities
Green fluorescent protein-based halide indicators with improved chloride and iodide affinities
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DOI：
10.1016/s0014-5793(01)02561-3
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发表时间：
2001-06-22
期刊：
影响因子：
3.5
通讯作者：
Verkman, AS
中科院分区：
文献类型：
--
作者：
Galietta, LJV;Haggie, PM;Verkman, AS
绿色荧光蛋白YFP-H148Q通过卤化物结合和pK(A)漂移的机制对卤化物敏感，但YFP-H148Q的局限性是其对卤素的低敏感性，对氯-的K-d>；为100 mm。对于细胞转运研究，特别是在通过高通量筛选发现药物以及测量亚细胞细胞器中的氯离子浓度方面，需要具有更高灵敏度的指示剂。在YFP-H148Q文库中，卤化物结合位点附近的一对残基被随机突变，开发了一种自动化程序来筛选细菌菌落，以提高对卤化物的敏感性。对1536个克隆的分析显示，阴离子敏感性得到改善，I-(I152L)的K-d降至2 mM，Cl-(V163S)的K-d降至40 mM，NO3-(I152L)的K-d降至10 mM。这些指示剂的阴离子敏感机制已被建立，并通过表达囊性纤维化跨膜电导调节因子氯通道的转基因细胞证明了它们在细胞中的有效性，(C)2001欧洲生化学会联合会。爱思唯尔科学公司出版。版权所有。
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 NO3- (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, (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.