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首页> 外文期刊>Biochemistry >Properties of recombinant fluorescent proteins from Photobacterium leiognathi and their interaction with luciferase intermediates.
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Properties of recombinant fluorescent proteins from Photobacterium leiognathi and their interaction with luciferase intermediates.

机译：来自Leiognathi细菌的重组荧光蛋白的性质及其与萤光素酶中间体的相互作用。

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摘要

Ligand binding and luciferase interaction properties of the recombinant protein corresponding to the lumazine protein gene (EMBL X56534) of Photobacterium leiognathi have been determined by fluorescence dynamics, circular dichroism, gel filtration, and SDS-PAGE. Scatchard analysis of a fluorescence titration shows that the apoprotein possess one binding site, and at 30 degrees C the KdS (microM) are as follows: 6,7-dimethyl-8-ribityllumazine, 0.26; riboflavin, 0.53; and much more weakly bound FMN, 30. All holoproteins are highly fluorescent and have absorption spectra distinct from each other and from the free ligands. The longest wavelength absorption maxima are, respectively (nm, 2 degrees C), 420, 463, and 458. Ligand binding produces no change in the far-UV circular dichroism; all have mean residual ellipticity at 210 nm of -6500 deg cm2 dmol-1, the same as the native protein. However, in the bioluminescence reaction only the lumazine holoprotein shows a bioluminescence effect. Fluorescence emission anisotropy decay was used to establish that none of these holoproteins complexed with native luciferase and that the lumazine protein alone formed a 1:1 complex with the luciferase hydroxyflavin fluorescent transient and the luciferase peroxyflavin intermediates, revealed by a dominant channel of anisotropy loss, with rotational correlation time of 2.5 ns, and attributed to excitation transfer from the luciferase flavin donor to the acceptor, the lumazine ligand. The complex stability was sufficient to allow its isolation by FPLC gel filtration and verification by SDS-PAGE. These methods also confirmed the absence of interaction of the holoflavoproteins.
机译：已经通过荧光动力学，圆二色性，凝胶过滤和SDS-PAGE确定了与光黑细菌的lumazine蛋白基因（EMBL X56534）相对应的重组蛋白的配体结合和荧光素酶相互作用特性。荧光滴定的Scatchard分析表明载脂蛋白具有一个结合位点，并且在30℃下的KdS（microM）如下：6，7-二甲基-8-ribityllumazine，0.26。核黄素0.53; 30。所有的全蛋白都是高度荧光的，吸收光谱彼此之间以及与游离配体不同。最长的波长吸收最大值分别为（nm，2摄氏度），420、463和458。配体结合不会在远紫外圆二色性上产生任何变化。与天然蛋白一样，它们在210 nm处的平均残留椭圆度为-6500 deg cm2 dmol-1。然而，在生物发光反应中，仅鲁嗪全蛋白显示出生物发光作用。荧光发射各向异性衰减用于确定这些全蛋白均不与天然荧光素酶复合，并且单独的二嗪蛋白与荧光素酶羟基黄素荧光瞬变和荧光素酶过氧黄素中间体形成1：1的复合物，这主要由各向异性损失的主要通道揭示，旋转相关时间为2.5 ns，归因于从萤光素酶黄素供体到受体lumazine配体的激发转移。复杂的稳定性足以使其通过FPLC凝胶过滤分离，并通过SDS-PAGE验证。这些方法还证实了全黄素蛋白不存在相互作用。

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