Heat perturbation of bovine eye lens α-crystallin probed by covalently attached ratiometric fluorescent dye 4′-diethylamino-3-hydroxyflavone

被引:12
作者
Avilov, SV
Bode, C
Tolgyesi, FG
Klymchenko, AS
Fidy, J
Demchenko, AR
机构
[1] AV Palladin Biochem Inst, Kiev, Ukraine
[2] Semmelweis Univ, Inst Biophys & Radiat Biol, H-1085 Budapest, Hungary
[3] Univ Louis Pasteur Strasbourg 1, Fac Pharm, CNRS, UMR 7034,Lab Pharmacol & Physicochim, F-67401 Illkirch Graffenstaden, France
[4] TUBITAK Res Inst Genet Engn & Biotechnol, TR-41470 Gebze, Turkey
关键词
6-bromomethyl-4 '-diethylamino-3-hydroxyflavone; eye lens alpha-crystallin; ratiometric fluorescence probe; small heat-shock protein; temperature perturbation;
D O I
10.1002/bip.20285
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Bovine eye lens a-crystallin was covalently labeled with 6-bromomethyl-4'-diethylamino-3-hydroxyflavone and studied under native-like conditions and at the elevated temperature (60 degrees C) that is known to facilitate alpha-crystallin chaperone-like activity. This novel SH-reactive two-band ratiometric fluorescent probe is characterized by two highly emissive N*- and T*-bands; the latter appears due to excited state intramolecular proton transfer reaction. The positions of these bands and the ratio of their intensities for the a-crystallin-dye conjugate are the sensitive indicators of polarity of the dye environment and its participation in intermolecular hydrogen bonding. Although we found that the dye labels both the SH and the NH2 groups in alpha-crystallin, a recently developed procedure allowed us to distinguish between the heat-induced spectral changes of the dye molecules attached to SH and NH2 groups. We observed that at elevated temperature the environment of the SH-attached dye becomes more polar and flexible. The number of H-bond acceptor groups in the vicinity of the dye decreases. Since alpha-crystallin contains a single Cys residue within the C-terminal domain of its alpha A subunit (the alpha B subunit contains none), we can attribute the observed effects to temperature-induced changes in the C-terminal domain of this protein. (c) 2005 Wiley Periodicals, Inc.
引用
收藏
页码:340 / 348
页数:9
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