EosFP,一种具有紫外线诱导的绿色到红色荧光转换功能的荧光标记蛋白。
EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.
作者信息
Wiedenmann Jörg, Ivanchenko Sergey, Oswald Franz, Schmitt Florian, Röcker Carlheinz, Salih Anya, Spindler Klaus-Dieter, Nienhaus G Ulrich
机构信息
Department of General Zoology and Endocrinology, University of Ulm, 89069 Ulm, Germany.
出版信息
Proc Natl Acad Sci U S A. 2004 Nov 9;101(45):15905-10. doi: 10.1073/pnas.0403668101. Epub 2004 Oct 25.
A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at approximately 390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Forster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.
一种来自石珊瑚赫氏叶状珊瑚(Lobophyllia hemprichii)的荧光蛋白基因已在大肠杆菌中克隆出来,并通过生化和生物物理方法进行了表征。我们将这种蛋白质命名为EosFP,它发出强烈的绿色荧光(516纳米),在约390纳米的近紫外光照射下会变为红色(581纳米),这是由于一种光诱导修饰,涉及发色团旁边肽主链的断裂。单分子荧光光谱表明,野生型EosFP是四聚体,各个荧光团之间存在强烈的福斯特共振耦合。我们分别通过引入单点突变V123T和T158H成功地将四聚体分解为AB和AC亚基二聚体,两种突变的组合产生了功能性单体。制备了与多种蛋白质的融合构建体并在人细胞中表达,表明其保留了正常的生物学功能。通过聚焦紫外光局部改变发射波长的可能性使EosFP成为用于追踪活细胞内生物分子运动实验的极佳标记物。