A genetically encoded fluorescent reporter of ATP:ADP ratio

被引：1

作者：

Berg, Jim ^{[1
]}

Hung, Yin Pun ^{[1
]}

Yellen, Gary ^{[1
]}

机构：

[1] Harvard Univ, Sch Med, Dept Neurobiol, Boston, MA 02115 USA

来源：

NATURE METHODS | 2009年 / 6卷 / 02期

基金：

美国国家卫生研究院;

关键词：

SENSITIVE K+ CHANNELS; ENERGY-CHARGE; BETA-CELLS; IN-VITRO; FREE ADP; PROTEINS; METABOLISM; KINASE; SENSOR; LIVER;

D O I：

10.1038/NMETH.1288

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

We constructed a fluorescent sensor of adenylate nucleotides by combining a circularly permuted variant of GFP with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The sensor's affinity for Mg-ATP was < 100 nM, as seen for other members of the bacterial PII regulator family, a surprisingly high affinity given that normal intracellular ATP concentration is in the millimolar range. ADP bound the same site of the sensor as Mg-ATP, competing with it, but produced a smaller change in fluorescence. At physiological ATP and ADP concentrations, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP: ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high-affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP: ADP ratio during live-cell imaging.

引用

页码：161 / 166

页数：6

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