Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.
Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.
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时间分辨室温蛋白质磷光：单发射色氨酸的非指数衰变。
DOI：
10.1016/s0006-3495(94)80588-0
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发表时间：
1994
影响因子：
3.4
通讯作者：
Gafni,A
中科院分区：
文献类型：
--
作者：
Schlyer,BD;Schauerte,JA;Steel,DG;Gafni,A
马肝醇脱氢酶（LADH）的单个室温磷光（RTP）残基。Trp-314和碱性磷酸酶（AP），Trp-109，显示非指数磷光衰减时，收集的数据，以高精度。使用最大熵方法（MEM）的分析，这些衰变，它表明，AP磷光衰减是由一个单一的高斯分布为主，而LADH的数据揭示了两个振幅包。两种蛋白质的MEM分布的寿命归一化宽度大于模型单指数发色团获得的宽度（例如，水溶液中的铽和环己烷中的芘）。实验表明，非指数衰减是基本的;即，这是纯蛋白质的内在属性。因为磷光报告了发光发色团的状态，这种非指数行为可能是由激发态反应的存在引起的。然而，众所周知，色氨酸残基的磷光寿命强烈依赖于吲哚部分周围的局部柔性。因此，非指数磷光衰减也可能是由对应于不同基态构象的至少两种不同局部刚性的状态（在磷光色氨酸附近）的存在引起的。在化学均匀的LADH样品中，磷光衰减动力学取决于激发波长的观察结果进一步支持了后一种解释。这种依赖性是由具有不同疏水性和刚性环境的LADH分子的子系综中Trp-314的波长选择性激发引起的。通过这种解释，数据表明，这些状态的相互转换发生在一个时间尺度长的磷光衰减（0.1-1.0秒）相比。进一步的实验表明，随着温度的升高，AP和LADH的分布磷光衰减速率变宽，从而表明1）在较高温度下填充的构象状态的数量增加或2）温度差异地影响单个构象状态。所观察到的异质三重态动力学的性质和它们之间的关系方面的蛋白质动力学进行了讨论。
The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1–1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.