Vincent M, Rouvière N, Gallay J
Laboratoire pour l'Utilisation du Rayonnement Electromagnétique (LURE), Centre National de la Recherche Scientifique, Centre Universitaire Paris-Sud, Orsay, France.
Cell Mol Biol (Noisy-le-grand). 2000 Sep;46(6):1113-31.
The time-resolved fluorescence intensity and anisotropy decays of the immunophilin domain of FKBP59 (FKBP59-I)--a protein containing two tryptophan residues (the W89, buried in a hydrophobic pocket and the W59, water exposed)--were studied using the time-correlated single photon counting (TCSPC) technique. The synchrotron radiation machine Super-ACO (Orsay, France) was used as a pulsed light source (approximately 8MHz). A mainly dual and discrete excited state lifetime distribution was previously evidenced (Rouvière et al., 1997). The lifetime heterogeneity has been suggested to be relevant to the topological tryptophan heterogeneity. Indeed, taking into account the spectroscopic properties of the single tryptophan residue of the immunophilin FKBP12, a highly homologous protein containing a single tryptophan residue, the short- and the long-lived lifetime species were assumed to be related to the solvent-buried and to the solvent-exposed fluorescent residues, respectively. We definitely demonstrate this point by describing the dynamical properties of each tryptophan residue of the FKBP59-I as a function of the emission wavelength. The data of the polarized components of the fluorescence emission were analyzed by the Maximum Entropy Method using a one-dimensional model (each excited-state lifetime tau being associated with each rotational correlation time theta) and a two-dimensional model (without any a priori association constraint between the tau's and the theta's). The two dimensional analysis of the polarized fluorescence intensity decays by MEM show the existence of a correlation between fast picosecond dynamics of the indole ring with the shortest-lived and blue emitting species. Conversely, the long-lived and red emitting population is mainly associated to the Brownian motion of the protein. A protein flexibility of the region located around the W59 residue, but slightly contributing to the light depolarization process, is also evidenced and can be specifically attributed to the red emitting population.
利用时间相关单光子计数（TCSPC）技术研究了FKBP59免疫亲和素结构域（FKBP59-I）的时间分辨荧光强度和各向异性衰减。FKBP59-I是一种含有两个色氨酸残基的蛋白质（W89埋藏在疏水口袋中，W59暴露于水中）。法国奥赛的同步辐射装置Super-ACO用作脉冲光源（约8MHz）。先前已证明存在主要为双重且离散的激发态寿命分布（鲁维耶等人，1997年）。寿命异质性被认为与拓扑色氨酸异质性有关。实际上，考虑到免疫亲和素FKBP12（一种含有单个色氨酸残基的高度同源蛋白质）的单个色氨酸残基的光谱特性，短寿命和长寿命物种分别被认为与溶剂埋藏和溶剂暴露的荧光残基有关。我们通过描述FKBP59-I每个色氨酸残基的动力学性质作为发射波长的函数，明确证明了这一点。荧光发射的偏振分量数据通过最大熵方法，使用一维模型（每个激发态寿命tau与每个旋转相关时间theta相关联）和二维模型（tau和theta之间没有任何先验关联约束）进行分析。通过MEM对偏振荧光强度衰减进行二维分析表明，吲哚环的快速皮秒动力学与最短寿命和蓝色发射物种之间存在相关性。相反，长寿命和红色发射群体主要与蛋白质的布朗运动有关。还证明了位于W59残基周围区域的蛋白质灵活性，但对光去极化过程的贡献较小，并且可以具体归因于红色发射群体。
