Ultrafast electron transfer in the complex between fluorescein and a cognate engineered lipocalin protein, a so-called anticalin
Ultrafast electron transfer in the complex between fluorescein and a cognate engineered lipocalin protein, a so-called anticalin
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DOI：
10.1021/bi015888y
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发表时间：
2002-03-26
期刊：
影响因子：
2.9
通讯作者：
Michel-Beyerle, ME
中科院分区：
文献类型：
--
作者：
Götz, M;Hess, S;Michel-Beyerle, ME
Anticalins是一类新的工程配体结合蛋白，具有来自脂质运载蛋白支架的定制特异性。安德灵FluA以高亲和力与荧光素配体络合，几乎完全淬灭其稳态荧光。为了研究潜在的机制，我们已经应用了铁秒吸收光谱，它揭示了激发态电子转移内的FluA（。）F1复合物对荧光有较强的猝灭作用。的氧化还原电位的比较的基础上，色氨酸或酪氨酸可以作为电子供体的结合荧光素基团在其激发单线态，从而形成荧光素三阴离子自由基内400 fs。几乎是单指数的速率指向一个单一的、明确定义的结合位点，并且其温度独立性表明（几乎）无活化过程。将传统的电子转移理论应用于超快的正向和较慢的反向速率，得到的电子相互作用相当大，酪氨酸的电子相互作用类似于140 cm（-1），这与在货车德瓦耳斯距离内两个芳香基团的共面排列一致。微弱的残余稳态荧光来源于时间常数在40 - 60 ps范围内的一个小组分（约10%）。这些结果表明，时间分辨吸收光谱作为一种诊断工具，用于阐明荧光猝灭机制和所涉及的过程的时间分布的权力。高结构和动态定义的络合位点表明anticalin RuA是一个很有前途的模型，以定制和探测蛋白质中的电子相互作用和能量。
Anticalins are a novel class of engineered ligand-binding proteins with tailored specificities derived from the lipocalin scaffold. The andealin FluA complexes fluorescein as ligand with high affinity, and it effects almost complete quenching of its steady-state fluorescence. To study the underlying mechanism, we have applied ferritosecond absorption spectroscopy, which revealed excited-state electron transfer within the FluA(.)Fl complex to be responsible for the strong fluorescence quenching. On the basis of a comparison of redox potentials, either tryptophan or tyrosine may serve as electron donor to the bound fluorescein group in its excited singlet state, thus forming the fluorescein trianion radical within 400 fs. The almost monoexponential rate points to a single, well-defined binding site, and its temperature independence suggests an (almost) activationless process. Applying conventional electron transfer theory to the ultrafast forward and slower back-rates, the resulting electronic interaction is rather large, with similar to140 cm(-1) for tyrosine, which would be consistent with a coplanar arrangement of both aromatic moieties within van der Waals distance. The weak residual steady-state fluorescence originates from a small (similar to10%) component with a time constant in the 40-60 ps range. These results demonstrate the power of timeresolved absorption spectroscopy as a diagnostic tool for the elucidation of a fluorescence quenching mechanism and the temporal profiles of the processes involved. The high structural and dynamic definition of the complexation site suggests the anticalin RuA to be a promising model in order to tailor and probe electronic interactions and energetics in proteins.