The emitting state of tryptophan in proteins with highly blue-shifted fluorescence
The emitting state of tryptophan in proteins with highly blue-shifted fluorescence
复制标题
DOI：
10.1002/anie.200700839
复制
发表时间：
2007-01-01
影响因子：
16.6
通讯作者：
Callis, Patrik R.
中科院分区：
文献类型：
--
作者：
Broos, Jaap;Tveen-Jensen, Karina;Callis, Patrik R.
色氨酸（Trp）的吲哚侧链的吸收带，在约280 nm处，包括两个重叠的过渡到1 La和1 Lb激发态。与基态相比，1 La的偶极矩较大，使得Trp荧光对微环境的变化具有高灵敏度，这反映在斯托克斯位移的范围内。[1]相比之下，1 Lb的偶极矩很小，与基态的偶极矩相当。鉴于这些状态的性质的差异，蛋白质的荧光数据只能在发射状态已知的情况下才能正确解释。[1]对于固态氩中的3-甲基吲哚（3 MI），1 Lb 0-0跃迁的能量比1 La跃迁低250 cm ↑ [2]，1 Lb是类似于气相或溶解在氟化烃中时的发射状态。[1b]在环己烷中，近简并的1 La和1 Lb的0-0跃迁观察。[1b]在更极性的有机溶剂或水中，发射来自1 La，因为这种状态对溶剂弛豫比1 Lb更敏感，并且1 La和1 Lb之间的激发态转换非常快。[3]对于一个色氨酸残基埋在一个蛋白质与烃侧链占主导地位的局部环境，远程静电相互作用的净效应，原则上可能会导致在1 La或1 Lb的发射状态。人们普遍认为色氨酸残基在1 La中发射出长波长荧光（320- 350 nm），但短波长荧光（% 310 nm）则存在争议。[1b在此，我们确定了天青蛋白、转氢酶dI蛋白以及新分离的dI突变体中短波长Trp的发射状态，该突变体具有迄今为止所描述的任何蛋白中最短波长的Trp发射。在这三种蛋白质中，只有最后一种产生了1 Lb发射的证据。铜绿假单胞菌的天青蛋白含有一个单一的Trp残基，是以前报道的在最短蓝移波长处发光的蛋白质。[4]最大发射波长在306- 308 nm，发射带为振动精细结构。[4]由于这些特征，它的发射通常被分配给1 Lb状态。[1b由于1 La和1 Lb态的跃迁偶极矩几乎相互垂直，各向异性激发谱可以决定性地确定哪个态发射。[6]在图1中，给出了全天青和中性Trp类似物N-乙酰基-色氨酸酰胺（NATA）在聚丙二醇（PG）玻璃中在1558 ℃下的激发光谱。0-0 1 Lb跃迁相对较强且尖锐[1b]，并且在291 nm处明显为肩部。如果1 Lb是发射状态，则该波长下的激发应导致高本征各向异性（r 0）。然而，对于载脂蛋白（未显示）和全天青蛋白，在1 Lb吸收处观察到r 0的急剧下降。[7]当吲哚环完全暴露于极性环境时，该模式与发射1 La的NATA [1b]记录的模式基本相同（图1a）。由于r 0几乎与发射波长无关，因此未获得混合发射的证据（图1b、d）。因此，这些数据与先前关于天青荧光发射来自1 Lb的建议不一致。在158 ℃下进行的实验，由于Trp侧链的旋转迁移率和蛋白质旋转预期会导致一些去极化，也导致r 0对λex的依赖性，这清楚地表明发射来自1 La（参见支持信息中的图S1）。F110 S天青蛋白突变体[4c]的各向异性激发光谱（其Trp残基经历独特的刚性局部环境[8]）也是相似的（图1 e，f）。
The absorption band of the indole side chain of tryptophan (Trp), at approximately 280 nm, comprises two overlapping transitions to the 1La and 1Lb excited states. Compared with the ground state, the dipole moment of 1La is large, giving Trp fluorescence its high sensitivity to changes in the microenvironment, as reflected in a range of Stokes shifts.[1] In contrast, the dipole moment of 1Lb is small and comparable with that in the ground state. Given the differences in the properties of these states, fluorescence data for proteins can only be properly interpreted if the emitting state is known.[1] For 3-methylindole (3MI) in solid argon, the 1Lb 0–0 transition is 250 cmÀ1 lower in energy than the 1La transition,[2] and 1Lb is the emitting state similar to when it is in the gas phase or when dissolved in a fluorinated hydrocarbon.[1b] In cyclohexane, near degeneracy of the 1La and 1Lb 0–0 transitions was observed.[1b] In more-polar organic solvents or in water, emission is from 1La because this state is much more sensitive to solvent relaxation than 1Lb, and excitedstate conversion between 1La and 1Lb is extremely fast.[3] For a Trp residue buried in a protein with a local environment dominated by hydrocarbon side chains, the net effect of longrange electrostatic interactions could, in principle, result in either 1La or 1Lb being the emitting state. There is a consensus view that Trp residues with long-wavelength fluorescence (320–350nm) emit from 1La, but short-wavelength Trp emission (% 310nm) is more contentious.[1b, c] Herein, we determine the emitting state of the short-wavelength Trp in azurin, in a dI protein from transhydrogenase and, notably, in a newly isolated mutant of dI, which has the shortestwavelength Trp emission of any protein described to date. Of the three proteins, only the last yields evidence of 1Lb emission.Azurin from Pseudomonas aeruginosa harbors a single Trp residue and is the protein that emits light at the shortest blue-shifted wavelength that has been previously reported.[4] The emission maximum (λmax) is at 306–308nm and the emission band shows vibrational fine structure.[4] Because of these features, its emission has often been assigned to the 1Lb state.[1b, 5] As the transition dipole moments of the 1La and 1Lb states are almost perpendicular to each other, anisotropy excitation spectra can conclusively determine which state is emitting.[6] In Figure 1, the excitation spectra of holoazurin and of the neutral Trp analogue, N-acetyl-tryptophan amide (NATA), in polypropylene glycol (PG) glass at À558C are presented. The 0–0 1Lb transition is relatively strong and sharp [1b] and is evident as a shoulder at 291 nm. If 1Lb is the emitting state, excitation at this wavelength should result in a high intrinsic anisotropy (r0). However, for both apo (not shown) and holoazurin, a sharp dip in r0 is observed at the 1Lb absorption.[7] This pattern is essentially the same as that recorded with the 1La-emitting NATA [1b] when the indole ring is completely exposed to a polar environment (Figure 1 a). No evidence for mixed emission was obtained as r0 is nearly independent of emission wavelength (Figure 1b, d). The data are, therefore, inconsistent with previous suggestions that azurin fluorescence emission is from 1Lb. Experiments performed at 158C, where some depolarization owing to rotational mobility of the Trp side chain and protein rotation is expected, also led to a dependence of r0 on λex, which clearly suggests that emission is from 1La (see FigureS1 in the Supporting Information). Anisotropy excitation spectra of the F110S azurin mutant,[4c] whose Trp residue experiences a uniquely rigid local environment,[8] again were similar (Figure 1 e, f).