Fluorescent BODIPY-GTP analogs: Real-time measurement of nucleotide binding to G proteins
Fluorescent BODIPY-GTP analogs: Real-time measurement of nucleotide binding to G proteins
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DOI：
10.1006/abio.2001.5011
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发表时间：
2001-04-01
影响因子：
2.9
通讯作者：
Neubig, RR
中科院分区：
文献类型：
--
作者：
McEwen, DP;Gee, KR;Neubig, RR
合成了三种BODIPY GTP γ S类似物（FL、515和TR）、BODIPY FL GppNHp和BODIPY FL GTP分子作为可能的荧光探针，以光谱学方式研究鸟嘌呤核苷酸结合。与G（α-微粒子）结合分别使基线类似物荧光增加6倍、8.5倍、2.8倍、3.5倍和3.0倍。GTP γ S和GppNHp类似物与G（α-微粒子）的结合具有高亲和力（K-D，分别为11、17、55和110 nM），并达到稳定的平台期，而BODIPY FL GTP的荧光显示短暂增加，返回基线。此外，BODIPY FL GTP gammaS对α（omicron）、α（s）、α（i1）和α（i2）显示不同的亲和力（6、58、150和300 nM）。BODIPY FL GppNHp对所有四个G（α）亚基的亲和力比BODIPY FL GTP γ S低10倍。荧光增加的半衰期与这些蛋白质的已知GDP释放速率一致。结合G（α）亚基后荧光的增强最可能是由于围绕γ-巯基（GTP γ S）或3 '核糖-羟基（GppNHp）键的旋转以减轻鸟嘌呤碱基对BODIPY荧光的淬灭。与G（α）结合使BODIPY部分暴露于外部环境，如通过碘化钠淬灭的增加所见。这些探针的可见激发和发射光谱以及高荧光水平允许对核苷酸结合进行鲁棒的实时检测。(C)北京：科学出版社.
Three BODIPY GTP gammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP molecules were synthesized as possible fluorescent probes to study guanine nucleotide binding spectroscopically. Binding to G(alpha omicron) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-fold, respectively. Binding of GTP gammaS and GppNHp analogs to G(alpha omicron) is of high affinity (K-D, 11, 17, 55, and 110 nM, respectively) and reaches a stable plateau while fluorescence of BODIPY FL GTP shows a transient increase which returns to baseline. Furthermore, BODIPY FL GTP gammaS shows varying affinities for alpha (omicron), alpha (s), alpha (i1), and alpha (i2) (6, 58, 150, and 300 nM). The affinities of BODIPY FL GppNHp for all four G(alpha) subunits are 10-fold lower than for BODIPY FL GTP gammaS. Half-times for the fluorescence increase are consistent with known GDP release rates for those proteins. Enhancement of fluorescence upon binding the G(alpha) subunit is most likely due to a rotation around the y-thiol (GTP gammaS) or the 3 ' ribose-hydroxyl (GppNHp) bond to relieve the quenching of BODIPY fluorescence by the guanine base. Binding to G(alpha) exposes the BODIPY moiety to the external environment, as seen by an increase in sodium iodide quenching. The visible excitation and emission spectra and high fluorescence levels of these probes permit robust real-time detection of nucleotide binding. (C) 2001 Academic Press.