RRC ID 50317
Author Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A.
Title Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer.
Journal Biochem J
Abstract GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) technology has facilitated the exploration of the spatio-temporal patterns of cellular signalling. While most studies have used cyan- and yellow-emitting FPs (fluorescent proteins) as FRET donors and acceptors respectively, this pair of proteins suffers from problems of pH-sensitivity and bleeding between channels. In the present paper, we demonstrate the use of an alternative additional donor/acceptor pair. We have cloned two genes encoding FPs from stony corals. We isolated a cyan-emitting FP from Acropara sp., whose tentacles exhibit cyan coloration. Similar to GFP from Renilla reniformis, the cyan FP forms a tight dimeric complex. We also discovered an orange-emitting FP from Fungia concinna. As the orange FP exists in a complex oligomeric structure, we converted this protein into a monomeric form through the introduction of three amino acid substitutions, recently reported to be effective for converting DsRed into a monomer (Clontech). We used the cyan FP and monomeric orange FP as a donor/acceptor pair to monitor the activity of caspase 3 during apoptosis. Due to the close spectral overlap of the donor emission and acceptor absorption (a large Förster distance), substantial pH-resistance of the donor fluorescence quantum yield and the acceptor absorbance, as well as good separation of the donor and acceptor signals, the new pair can be used for more effective quantitative FRET imaging.
Volume 381(Pt 1)
Pages 307-12
Published 2004-7-1
DOI 10.1042/BJ20040321
PII BJ20040321
PMID 15065984
PMC PMC1133789
MeSH Amino Acid Sequence Amino Acids / chemistry Amino Acids / genetics Animals Anthozoa / chemistry Bacterial Proteins / chemistry Bacterial Proteins / metabolism Caspase 3 Caspases / metabolism Fluorescence Resonance Energy Transfer / classification Fluorescence Resonance Energy Transfer / methods* Fluorescent Dyes / chemistry Fluorescent Dyes / metabolism Green Fluorescent Proteins Hydrogen-Ion Concentration Luminescent Proteins / chemistry Luminescent Proteins / genetics Luminescent Proteins / metabolism* Molecular Sequence Data Mutagenesis, Site-Directed / genetics
IF 4.331
Times Cited 237
Resource
DNA material MiCy/pRSETB (RDB15239) MiCy-DEVD-mKO1/pCS2 (RDB15240) mMiCy1/pRSETB (RDB15241) KO1/pRSETB (RDB15311) hmKO1/pRSETB (RDB15312) hmKO2/pRSETB (RDB15313).