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首页> 外文期刊>Biochemistry >Cyan Fluorescent Protein Carries a Constitutive Mutation That Prevents Its Dimerization
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Cyan Fluorescent Protein Carries a Constitutive Mutation That Prevents Its Dimerization

机译:青色荧光蛋白携带一个组成型突变,可防止其二聚化

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The tendency of G FP-like fluorescent proteins to dimerize in vitro is a permanent concern as it may lead to artifacts in FRET imaging applications. However, we have found recently that CFP and YFP (the couple of GFP variants mostly used in FRET studies) show no trace of association in the cytosol of living cells up to millimolar concentrations. In this study, we investigated the oligomerization properties of purified CFP, by fluorescence anisotropy and sedimentation velocity. Surprisingly, we found that CFP has a much weaker homoaffinity than other fluorescent proteins (K_d >= 3 x 10~(-3) M), and that this is due to the constitutive N146I mutation, originally introduced into CFP to improve its brightness.
机译:G FP样荧光蛋白在体外二聚化的趋势是一个永久性的问题,因为它可能会在FRET成像应用中导致伪影。但是,我们最近发现,CFP和YFP(在FRET研究中最常使用的GFP变体对)在毫摩尔浓度以下的活细胞的胞质溶胶中均未发现任何关联。在这项研究中,我们通过荧光各向异性和沉降速度研究了纯化CFP的低聚特性。出乎意料的是,我们发现CFP的同源性比其他荧光蛋白(K_d> = 3 x 10〜(-3)M)弱得多,这是由于N146I组成性突变最初引入CFP中以提高其亮度。

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