Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China.
J Biol Inorg Chem. 2010 Sep;15(7):995-1007. doi: 10.1007/s00775-010-0660-z. Epub 2010 Apr 29.
Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins. It has been proven, using Tb3+ as a fluorescence probe, that EoCen has four calcium-binding sites. The sensitized emission arises from nonradiative energy transfer between the three tyrosine residues (Tyr46, Tyr72, and Tyr79) of the N-terminal half and the bound Tb3+ ions. To determine the most critical of the three tyrosine residues for the process of fluorescence resonance energy transfer, six mutants of the N-terminal domain of EoCen, which contain one (N-Tyr46/N-Tyr72/N-Tyr79) or two (N-Y46F/N-Y72F/N-Y79F) tyrosine residues, were obtained by site-directed mutagenesis. The aromatic residue-sensitized Tb3+ fluorescence of N-Y79F was most affected, displaying a 50% reduction compared with wild-type N-EoCen. Among the tyrosines, Tyr79 is the shortest mean distance from the protein-bound Tb3+ (at sites I/II), as calculated via the Förster mechanism. The steady-state and time-resolved fluorescence parameters of the wild-type N-EoCen and the three double mutants suggest that Tyr79, which exists in a hydrophobic environment, has the highest quantum yield and a relatively long average lifetime. The decay of Tyr79 is the least heterogeneous among the three tyrosine residues. In addition, molecular modeling shows that a critical hydrogen bond is formed between the 4-hydroxyl group of Tyr79 and the oxygen from the side chains of the residue Asn39. Kinetic experiments on tyrosine and Tb3+ fluorescence demonstrate that tyrosine fluorescence quenching is largely due to the self-assembly of EoCen, and that the quenching degrees of the mutants differ. Resonance light scattering and crosslinking analysis carried out on the full-length single mutants (Y46F, Y72F, and Y79F) showed that Tyr79 also plays the most important role in the Tb3+-dependent self-assembly of EoCen among the three tyrosines.
纤毛虫八肋游仆虫中心体蛋白（EoCen）是钙结合蛋白 EF 手超家族的成员。已经证明，使用 Tb3+作为荧光探针，EoCen 有四个钙结合位点。敏化发射是由 N 端半胱氨酸的三个酪氨酸残基（Tyr46、Tyr72 和 Tyr79）与结合的 Tb3+离子之间的非辐射能量转移引起的。为了确定三个酪氨酸残基中对荧光共振能量转移过程最关键的一个，通过定点突变获得了 EoCen N 端结构域的六个突变体，其中包含一个（N-Tyr46/N-Tyr72/N-Tyr79）或两个（N-Y46F/N-Y72F/N-Y79F）酪氨酸残基。与野生型 N-EoCen 相比，N-Y79F 的芳香族残基敏化 Tb3+荧光受到的影响最大，显示出 50%的降低。在这些酪氨酸中，Tyr79 与蛋白结合的 Tb3+（在位点 I/II）的平均距离最短，这是通过福斯特机制计算出来的。野生型 N-EoCen 和三个双突变体的稳态和时间分辨荧光参数表明，存在于疏水环境中的 Tyr79 具有最高的量子产率和相对较长的平均寿命。三个酪氨酸残基中，Tyr79 的衰减最不均匀。此外，分子建模表明 Tyr79 的 4-羟基与残基 Asn39 的侧链氧之间形成了一个关键氢键。酪氨酸和 Tb3+荧光的动力学实验表明，酪氨酸荧光猝灭主要归因于 EoCen 的自组装，并且突变体的猝灭程度不同。对全长单突变体（Y46F、Y72F 和 Y79F）进行的共振光散射和交联分析表明，在三个酪氨酸中，Tyr79 也在 EoCen 依赖于 Tb3+的自组装中起着最重要的作用。
