Effects of the Disappearance of One Charge on Ultrafast Fluorescence Dynamics of the FMN Binding Protein
Effects of the Disappearance of One Charge on Ultrafast Fluorescence Dynamics of the FMN Binding Protein
复制标题
DOI：
10.1021/jp912137s
复制
发表时间：
2010-05-13
影响因子：
3.3
通讯作者：
Tanaka, Fumio
中科院分区：
文献类型：
--
作者：
Chosrowjan, Haik;Taniguchi, Seiji;Tanaka, Fumio
用x射线衍射法测定了Miyazaki F株Desulfovibrio vulgaris中的E13T （G1u13被Thr13取代）和E13Q （G1u13被G1n13取代）FMN结合蛋白（FMN-bp）的晶体结构。比较了三种形式的FMN-bp光导电子从Trp32、Tyr35和Trp106向受激异alloxazine （Iso*）转移的几何因素。在FMN-bp中，从Trp32到Iso*的ET速率被认为是最快的，其次是Tyr35和Trp106。在wr、Elyr和E13Q的FiV1Nbps中，lso与Trp32之间的距离没有明显变化（0.705 ~ 0.712 nm），而Iso与Tyr35或Trp106之间的距离在两个突变的FMN-bps中都缩短了约0.01 nm。然而，与WT相比，突变蛋白中13位残基与ET供体或受体之间的距离发生了显著变化。当Glu 13被Thr13或Gin 13取代时，氢键对和Is与周围氨基酸之间的距离没有改变。采用荧光上转换的方法，对比研究了Glu 13离子电荷消除对E13T和E13Q超快荧光动力学的影响。荧光寿命在El3T中分别为tau(1) = 107 fs （alpha(1) = 0.86）、tau(2)= 475 fs （alpha(2) = 0.12）、tau(3) = 30 Ps (alpha(3) = 0.02)， E13Q中分别为tau(1) = 134 fs （alpha(1) = 0.85）、tau(2)= 746 fs （alpha(2) = 0.12）、tau(3) = 30 Ps (alpha(3) = 0.03)，与WT中报道的tau(1) = 168 fs （alpha(1) = 0.95）、tau(2)= 1.4 Ps （alpha(2) = 0.05）进行比较。平均寿命(tau(Av) = Sigma(2or3)(i=1)alpha（i)iota(o)）在E13T中为0.75 ps，在E13Q中为1.10 ps，在WT中为0.23 ps，这意味着tau（Av）在E13T中比WT长3.3倍，在E13Q中比WT长4.8倍。当溶剂由H2O变为D2O时，WT的超快荧光动力学没有改变。用FMN-bp三种体系的静态结构分析了静态Er率（平均寿命的倒数）。ET速率依赖于Iso和Trp32的净静电能（ES）， WT为0.0263 eV， E13T为0.322 eV， E13Q为0.412 eV。计算的蒸散发速率与观测的蒸散发速率非常吻合。
Crystal structures of E13T (G1u13 was replaced by Thr13) and E13Q (G1u13 was replaced by G1n13) FMN binding proteins (FMN-bp) from Desulfovibrio vulgaris, strain Miyazaki F, were determined by the X-ray diffraction method. Geometrical factors related to photoinduced electron transfer from Trp32, Tyr35, and Trp106 to the excited isoalloxazine (Iso*) were compared among the three forms of FMN-bp. The rate of ET is considered to be fastest from Trp32 to Iso* in FMN-bp and then from Tyr35 and Trp106. The distances between lso and Trp32 did not change appreciably (0.705-0.712 nm) among wr, Elyr, and E13Q FiV1Nbps, though the distances between Iso and Tyr35 or Trp106 became a little shorter by ca. 0.01 nm in both mutated FMN-bps. The distances between the residue at 13 and the ET donors or acceptor in the mutated proteins, however, changed markedly, compared to WT. Hydrogen bonding pairs and distances between Is and surrounding amino acids were not modified when Glu 13 was replaced by Thr13 or Gin 13. Effects of elimination of ionic charge at Glu 13 on the ultrafast fluorescence dynamics in E13T and E13Q were investigated comparing to WT, by means of a fluorescence up-conversion method. Fluorescence lifetimes were tau(1) = 107 fs (alpha(1) = 0.86), tau(2) = 475 fs (alpha(2) = 0.12), and tau(3) = 30 Ps (alpha(3) = 0.02) in El3T and tau(1) = 134 fs (alpha(1) = 0.85), tau(2)= 746 fs (alpha(2) = 0.12), and tau(3) = 30 ps (alpha(3) = 0.03) in E13Q, which are compared to the reported lifetimes in WT, tau(1) = 168 fs (alpha(1) = 0.95) and tau(2) = 1.4 Ps (alpha(2) = 0.05). Average lifetimes (tau(Av) = Sigma(2or3)(i=1)alpha(i)iota(o)) were 0.75 ps in E13T, 1.10 ps in E13Q, and 0.23 ps in WT, which implies that tau(AV) was 3.3 times longer in E13T and 4.8 times longer in E13Q, compared to WT. The ultrafast fluorescence dynamics of WT did not change when solvent changed from H2O to D2O. Static Er rates (inverse of average lifetimes) were analyzed with static structures of the three systems of FMN-bp. Net electrostatic (ES) energies of Iso and Trp32, on which ET rates depend, were 0.0263 eV in WT, 0.322 eV in E13T, and 0.412 eV in E13Q. The calculated ET rates were in excellent agreement with the observed ones in all systems.