RRC ID 49218
Author Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A.
Title Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.
Journal Proc Natl Acad Sci U S A
Abstract Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.
Volume 101(29)
Pages 10554-9
Published 2004-7-20
DOI 10.1073/pnas.0400417101
PII 0400417101
PMID 15247428
PMC PMC490022
MeSH Animals Bacterial Proteins / chemistry* Bacterial Proteins / genetics Bacterial Proteins / metabolism* Calcium / metabolism* Fluorescence Resonance Energy Transfer / methods Fluorescent Dyes / metabolism* HeLa Cells Humans Luminescent Proteins / chemistry* Luminescent Proteins / genetics Luminescent Proteins / metabolism* Mice Microscopy, Fluorescence / methods Models, Molecular Molecular Sequence Data Protein Conformation*
IF 9.412
Times Cited 772
DNA material YC2.12/pCS2 (RDB15132) YC3.12/pCS2 (RDB15133) YC2.60/pRSETB (RDB15134) YC3.60/pcDNA3 (RDB15135) YC3.60/pRSETB (RDB15136) YC3.60pm/pRSETB (RDB15137) YC4.60/pRSETB (RDB15138) cp49Venus (RDB15120) cp145Venus (RDB15121) cp157Venus (RDB15122) cp173Venus (RDB15123) cp195Venus (RDB15124) cp229Venus (RDB15125).