Time-resolved single tryptophan fluorescence in photoactive yellow protein monitors changes in the chromophore structure during the photocycle via energy transfer.
Time-resolved single tryptophan fluorescence in photoactive yellow protein monitors changes in the chromophore structure during the photocycle via energy transfer.
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光敏黄色蛋白中的时间分辨单色氨酸荧光通过能量转移监测光循环期间发色团结构的变化。
DOI：
10.1021/bi051448l
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Heyn,MaartenP
中科院分区：
文献类型：
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作者：
Otto,Harald;Hoersch,Daniel;Meyer,TerryE;Cusanovich,MichaelA;Heyn,MaartenP
我们从时间分辨的荧光强度和去偏振实验表明，PYP的独特的色氨酸W119的荧光被淬灭的能量转移到4-羟基肉桂酰基发色团。而强度衰减的时间常数为0.18 ns的P，衰减的辅因子（apo-PYP）的情况下，有一个单一的指数寿命为4.8 ns。有和没有受体的这种寿命差异可以根据能量转移和P的高分辨率X射线结构定量解释，这允许精确计算κ 2因子。供体和受体的荧光去极化实验表明，两者都被固定，使得κ 2在荧光时间尺度上是恒定的。使用来自在470 nm处发射的LED的背景照明，我们测量了P和中间体I2和I2 '的光稳混合物中的时间分辨荧光。光稳定混合物的组成取决于pH，并且从低pH下的主要I2变为高pH下的主要I2'。I2/I2'平衡是pH依赖性的，pKa为1.63。在I2中，寿命增加到10.82 ns。这不是由于距离的变化或光谱重叠的增加，而是主要是κ2大幅下降的结果。κ 2是根据可用的X射线结构计算的，从P中的2.7降至I2中的0.27。κ 2的这种变化是由受体的异构化引起的，这导致其跃迁偶极矩的重新取向。我们在这里有一个罕见的情况下，κ 2因子主导的能量转移的变化。光中的荧光衰减是pH依赖性的。从SVD分析的光/暗差强度衰减在一些pH值，我们确定了三个物种与相关的寿命：P（0.18 ns），I2（0.82 ns），和X（0.04 ns）。基于与I2和X相关的振幅的pH依赖性，在pKa = 6.3的情况下，我们将第三种物质分配给信号状态I2 '。的0.82和0.04 ns的物种的吸收光谱计算从pH值的依赖性，它们的荧光振幅和光稳定的光/暗差吸收光谱。这些光谱（372和352 nm）的λ max值分别识别了I2和I2 '的0.82和0.04 ns分量，并验证了荧光数据分析。突变体E46 Q允许进一步测试能量转移的解释，因为在黑暗中降低pH值会导致漂白状态，光谱重叠增加，但没有异构化诱导的κ2降低。测得的寿命为0.04纳秒是在很好的协议与预测的基础上的能量转移和X射线结构。
We show from time-resolved fluorescence intensity and depolarization experiments that the fluorescence of the unique tryptophan W119 of PYP is quenched by energy transfer to the 4-hydroxycinnamoyl chromophore. Whereas the intensity decay has a time constant of 0.18 ns in P, the decay in the absence of the cofactor (apo-PYP) has a single exponential lifetime of 4.8 ns. This difference in lifetime with and without acceptor can be explained quantitatively on the basis of energy transfer and the high-resolution X-ray structure of P, which allows an accurate calculation of the κ2factor. Fluorescence depolarization experiments with donor and acceptor indicate that both are immobilized so that κ2is constant on the fluorescence time scale. Using background illumination from an LED emitting at 470 nm, we measured the time-resolved fluorescence in a photostationary mixture of P and the intermediates I2and I2‘. The composition of the photostationary mixture depends on pH and changes from mainly I2at low pH to predominantly I2‘ at high pH. The I2/I2‘ equilibrium is pH-dependent with a pKaof ∼6.3. In I2the lifetime increases to ∼0.82 ns. This is not due to a change in distance or to the increase in spectral overlap but is primarily a consequence of a large decrease in κ2. κ2was calculated from the available X-ray structures and decreases from ∼2.7 in P to 0.27 in I2. This change in κ2is caused by the isomerization of the acceptor, which leads to a reorientation of its transition dipole moment. We have here a rare case of the κ2factor dominating the change in energy transfer. The fluorescence decay in the light is pH-dependent. From an SVD analysis of the light/dark difference intensity decay at a number of pH values, we identify three species with associated lifetimes: P (0.18 ns), I2(0.82 ns), and X (0.04 ns). On the basis of the pH dependence of the amplitudes associated with I2and X, with a pKaof ∼6.3, we assign the third species to the signaling state I2‘. The absorption spectra of the 0.82 and 0.04 ns species were calculated from the pH dependence of their fluorescence amplitudes and of the photostationary light/dark difference absorption spectra. The λmaxvalues of these spectra (372 and 352 nm) identify the 0.82 and 0.04 ns components with I2and I2‘, respectively, and validate the fluorescence data analysis. The mutant E46Q allows a further test of the energy transfer explanation, since lowering the pH in the dark leads to a bleached state with an increased spectral overlap but without the isomerization-induced decrease in κ2. The measured lifetime of 0.04 ns is in excellent agreement with predictions based on energy transfer and the X-ray structure.