Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F-NMR methodologies.
Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F-NMR methodologies.
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使用荧光和 19F-NMR 方法检查氟化铝结合位点的延伸因子 Tu。
DOI：
10.1016/0014-5793(91)80122-j
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发表时间：
1991
期刊：
影响因子：
3.5
通讯作者：
Jameson,DM
中科院分区：
文献类型：
--
作者：
Hazlett,TL;Higashijima,T;Jameson,DM
本文报道了AP 3+和F−与两种GTP结合蛋白，延伸因子Tu（EF-Tu）和激素敏感调节蛋白（G蛋白）Goα的相互作用的比较。选择用于阐明蛋白质和氟化铝之间可能的方法是荧光光谱法和核磁共振（19 F-NMR）。这两种蛋白质在其核苷酸结合位点附近都有色氨酸残基，这是氟化铝相互作用的假定位点。对于G蛋白（包括Goα），假设在Mg 2+存在下，氟化铝模拟镁配位的γ-磷酸基团形成GDP-形式的蛋白质，并使蛋白质的构象向活性GTP-形式转变。事实上，观察到氟化铝影响Goα的固有荧光的变化。氟化铝的存在并不影响EF-Tu·GDP的固有荧光、寿命谱。然后使用19 F-NMR直接测试结合F−。单独的氟化物或在任一种蛋白质的存在下，在−10 ppm处产生一个19 F-NMR峰，这是游离F−的特征。在蛋白质和F−样品中加入铝后，观察到Goα·GDP的第二个峰，从第一个峰上移到-29 ppm。这第二个峰被认为是蛋白结合F−，但在EF-Tu·GDP中没有观察到。这些观察结果表明，在Mg 2+存在的情况下，Al 3+和F−的相互作用在结合氟化铝的激素敏感性G蛋白和包括EF-Tu在内的整体GTP结合蛋白之间可能非常不同。因此，当延伸因子的结构数据，特别是核苷酸位点的结构数据用于解释数据或构建用于描述激素敏感性调节G蛋白的模型时，必须谨慎。
This article reports on a comparison or the interaction of AP3+and F−with two GTP‐binding proteins, elongation factor Tu (EF‐Tu) and the hormone sensitive regulatory protein (G protein) Goα. The methodologies chosen to elucidate possible between protein and aluminum fluoride were fluorescence spectroscopy and nuclear magnetic resonance (19F‐NMR). Both proteins have tryptophan residues near their nucleotide binding sites, the purported site of aluminum fluoride interaction. It has been assumed for G proteins (including Goα) that aluminum fluoride, in the presence of Mg2+, mimics the magnesium coordinated γ‐phosphate group for the GDP‐form of the protein and shifts the protein's conformation toward the active GTP‐form. Indeed, changes in intrinsic fluorescence of Goα affected by aluminum fluoride are observed. The presence of aluminum fluoride did not affect the intrinsic fluorescence, spectra of lifetimes, of EF‐Tu·GDP.19F‐NMR was then used to directly test for bound F−. Fluoride alone or in the presence of either protein gave a single19F‐NMR peak at −10 ppm, characteristic of free F−. With the addition of aluminum to the protein and F−samples a second peak, shifted upfield from the first to −29 ppm, was observed for Goα·GDP. This second peak, which has been assigned to protein‐bound F−, was not observed for EF‐Tu·GDP. These observations show that the interaction of Al3+and F−, in the presence of Mg2+, may be quite different between the hormone‐sensitive G proteins, which bind aluminum fluoride, and the GTP‐binding proteins as a whole, which include EF‐Tu. Care must therefore be exercised when structural data on the elongation factor, specifically on the nucleotide site, are used to interpret data or compose models intended to describe the hormone‐sensitive regulatory G proteins.