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A novel analytical method for in vivo phosphate tracking
Author(s) -
Gu Hong,
Lalonde Sylvie,
Okumoto Sakiko,
Looger Loren L.,
Scharff-Poulsen Anne Marie,
Grossman Arthur R.,
Kossmann Jens,
Jakobsen Iver,
Frommer Wolf B.
Publication year - 2006
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2006.09.048
Subject(s) - förster resonance energy transfer , chemistry , biophysics , fluorescence , phosphate , biochemistry , transporter , biology , gene , physics , quantum mechanics
Genetically‐encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (P i ) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate‐binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows P i ‐dependent increases in FRET efficiency. FLIPPi affinity mutants cover P i changes over eight orders of magnitude. COS‐7 cells co‐expressing a low‐affinity FLIPPi and a Na + /P i co‐transporter exhibited FRET changes when perfused with 100 μM P i , demonstrating concentrative P i uptake by PiT2. FLIPPi sensors are suitable for real‐time monitoring of P i metabolism in living cells, providing a new tool for fluxomics, analysis of pathophysiology or changes of P i during cell migration.