980 Prog Biochem Biophys FRET 2 3 33 (, 510631) FRET 2,, 2,, 2 (fluorescence resonance energy transfer, FRET),, 2, ( FRET), 2, Q6 ( ) GFP CFP YFP : CFP : ( fluorescence YFP, resonance energy transfer, FRET),, CFP, CFP YFP 2, CFP, (green fluorescent protein, GFP) YFP GFP 6 [1,2,,, ] GFP, a b, FRET,, GFP,, : CFP byfp, GFP 433 nm, a b 2, CFP YFP GFP,, 476 nm, CFP ; a b,, ( yellow b a, fluorescent protein, YFP), ( cyan CFP YFP, fluorescent protein, CFP ) 527 nm YFP FRET 2 (1) 1 FRET,,,, CFP, 2 3 (2002CCC00400) (015012) 33 Tel : 020285210089, E2mail : xingda @scnu edu cn : 2003204204, : 2003205228 1995-2005 Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved
Prog Biochem Biophys 981 Fig11 FRET principle 1 FRET FRET : CFP (cyan fluorescent protein) byfp ( yellow fluorescent protein) CFP 433nm : 1 CFP YFP, CFP 476 nm ; 2, CFPYFP (fluorescence resonance energy transfer, FRET), 527 nm YFP, 2 2 FRET,, 2,,, [3,4, 2 ] 4, : PKA (protein kinase A) Src : AblEGFR (epidermal growth factor receptor), FRET,, 2, 25 %35 % FRET forskolin PKA FRET, 25 %50 %, FRET 211 GFP CFP YFP, FRET,, FRET, PKA 21111 :, FRET,,, 21112 :,,,, ;, FRET, ; Zhang [3 ] FRET,, ( ), (cysteinyl aspartate2specific protease, Caspase), 1995-2005 Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved
982 Prog Biochem Biophys FRET, FRET Reiko [5 ] Lyn FRET Caspase8 Bid, myristoyl palmitoyl CFP, Caspase8 Bid, YFP FRET,, c Bid MCD (52methyl2 2cyclodextrin) CFP YFP,, FRET, Bid FRET, Caspase8, Bid CFP YFP,, CFP,,, CFP, Fas Markus [6 ] Kiwamu [7 ] FRET Caspase3 [10,11 ],,,, Caspase3 Fas FRET, 212 21211 : Fas Rocheville [12,13 ], SSTR5 ( type 5 somatostatin receptor), D2R (type 2 dopamine receptor)? Peter [8 ],,,, SSTR5 EGFR (epidermal growt h factor receptor) GFP, EGFR SSTR5, D2R SSTR5 Cy3, EGF ( epidermal 21213 : TNFR ( ) Fas CFPYFP, somatostatin,, growth factor) Cy5,?, EGF EGF FRET ( SSTR5, D2R, EGFR, GFP ) Cy3, FRET FRET,, 213 21212 : Rho G, (lipid, rafts) (caveolae),, GTPase,?? Zacharias [9 ], Kraynov [14 ] FLAIR (fluorescence activation indicator for Rho proteins) FRET, GFP CFP YFP PA K1, Rac2GTP domain PDB Alexa, GFP Rac,, Rac PDB, GFP Alexa, CFP YFP FRET 1995-2005 Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved
Prog Biochem Biophys 983 Rac Rac Matsuda [15 ] Ras Rap1, FRET Ras Raf Ras ( Raf RBD) GFP YFP CFP Ras YFP, Raf RBD CFP,, FRET GEFs ( guanine2nucleotide exchange factors) GAPs ( GTPase2activating proteins), FRET Ras Rap1 3 FRET,,,,, FRET, 1 Pollok B A, Heim R Using GFP in FRET2based applications Trends Cell Biol, 1999, 9 (2) : 5760 2 Lakowicz J R Principles of Fluorescence Spectroscopy New York : Plenum Press, 1983 305337 3 Zhang J, Ma Y L, Taylor S S, et al Genetically encoded reporters of protein kinase A activity reveal impact of substrate tethering Proc Natl Acad Sci USA, 2001, 98 ( 26) : 14997 15002 4 Ting A Y, Kain K H, Klemke R L, et al Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells Proc Natl Acad Sci USA, 2001, 98 (26) : 1500315008 5 Reiko O, Nagasaki A, Kawasaki H, et al Confirmation by FRET in individual living cells of the absence of significant amyloid 2 mediated caspase 8 activation Proc Natl Acad Sci USA, 2002, 99 (23) : 1471614721 6 Markus R, Dussmann H, Janicke R U, et al Single2cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process J Biol Chem, 2002, 277 (27) : 2450624514 7 Kiwamu T, Nagai T, Miyawaki A, et al Spatio2temporal activation of caspase revealed by indicator that is insensitive to environmental effects J Cell Biol, 2003, 160 (2) : 235243 8 Peter J, Wouters F S, Reynolds A R, et al Quantitative imaging of lateral ErbB1 receptor signal propagation in the plasma membrane Science, 2000, 290 (5496) : 15671570 9 Zacharias D A, Violin J D, Newton A C, et al Partitioning of lipid2modified monomeric GFPs into membrane microdomains of live cells Science, 2002, 296 (5569) : 913916 10 Richard M S, John K F, Zacharias D A, et al Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations Science, 2000, 288 (5475) : 23542357 11 Francis K M C, Hyung J C, Zheng L, et al A domain in TNF receptors that mediates ligand2independent receptor assembly and signaling Science, 2000, 288 (5475) : 23512354 12 Rocheville M, Lange D C, Kumar U, et al Receptors for dopamine and somatostatin : formation of hetero2oligomers with enhanced functional activity Science, 2000, 288 (5463) : 154 157 13 Rocheville M, Lange D C, Kumar U, et al Subtypes of the somatostatin receptor assemble as functional homo2 and heterodimers J Biol Chem, 2000, 275 (11) : 78627869 14 Kraynov V S, Chamberlain C, Bokoch G M, et al Localized Rac activation dynamics visualized in living cells Science, 2000, 290 (5490) : 333337 15 Mochizuki N, Yamashita S, Kurokawa K, et al Spatio2temporal images of growth2factor2induced activation of Ras and Rap1 Nature, 2001, 411 (6841) : 10651068 Fluorescence Resonance Energy Transfer Assay : Applications in The Study of Protein2protein Interaction 3 WAN G Jin2J un, CHEN Xiao2Chuan, XIN G Da 33 ( Instit ute of L aser L if e Science, South Chi na Normal U niversity, Guangz hou 510631, China) Abstract Protein2protein interactions play a central role in numerous processes in the cell Given the complex organization of cell, it is very likely that the molecular behavior of proteins in a test tube is different from that in living cells For t his reason, t he conventional approaches used to st udy protein2protein interactions, including two2hybrid yeast assay, immunoprecipitation assay, etc, may not represent t he p hysiological nat ure in living cells Fluorescence resonance energy t ransfer ( FRET) is a recently developed laser technique, which offers a unique opport unity to monitor real2time protein2protein interactions or protein conformational changes wit hin a living cell and to follow t he time course of t he changes in FRET corresponding to cellular event s The observation of such dynamic molecular event s in it s nat ural environment provides vital insight into t he protein2 1995-2005 Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved
984 Prog Biochem Biophys protein interactions wit hin t he cell The applications of t his approach to st udy t he protein2protein interactions i n vivo are briefly reviewed Key words fluorescence resonance energy t ransfer ( FRET), protein2protein interactions, living cell 3 This work was supported by grants from The National Major Fundamental Research Project of China (2002CCC00400) and The Team Project of Natural Science Foundation of Guangdong Province (015012) 33 Corresponding author Tel : 86220285210089, E2mail : xingda @scnu edu cn Received : April 4, 2003 Accepted : May 28, 2003 2004 5 1517, 2004 2 15, :, : 010264888485 ; 0102 64888489 : 010264872026 E2mail : tyl @sun5 ibp ac cn ;, nlboff @sun5 ibp ac cn 5 1819,, :, :,,, - - - - - - - - - - - - - - - - - - - - - - - - - 2004 5 1517, A B, /,, ; ; ; ;, 5 14, 5 1516, 5 17, 5 17 E2mail 600 /, 300 /, 10 / /,, : A4, 2154 cm, 3117 cm : : 4,, New Roman : A B ( ) / : 5,, : 4, : : 5, : 2004 4 20 (, ) 100101 15, E2mail 1995-2005 Tsinghua Tongfang Optical Disc Co, Ltd All rights reserved