RRC ID 49218
著者 Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A.
タイトル Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.
ジャーナル 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.
巻・号 101(29)
ページ 10554-9
公開日 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
引用数 772
WOS 分野 BIOCHEMISTRY & MOLECULAR BIOLOGY
リソース情報
遺伝子材料 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).