Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent.pdf

Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent.pdf

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ExpandeddynamicrangeoffluorescentindicatorsforCa2bycircularlypermutedyellowfluorescent

Expanded dynamic range of fluorescent indicators for Ca2 by circularly permuted yellow fluorescent proteins Takeharu Nagai*?, Shuichi Yamada?, Takashi Tominaga§, Michinori Ichikawa§, and Atsushi Miyawaki*? *Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, and §Laboratory for Brain-Operative Devices, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; ?Structure and Function of Biomolecules, Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 351-0198, Japan; and ?Laboratory of Signal Transduction, Institute for Virus Research, Kyoto University, Kawaharacho 53, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Edited by Roger Y. Tsien, University of California at San Diego, La Jolla, CA, and approved May 19, 2004 (received for review January 29, 2004) Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for vari- ous 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 Ca2 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 Ca2-saturated form, thereby increasing the Ca2-dependent change in the ratio of YFPCFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca2 dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-

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