van Thor Jasper J, Zanetti Giulia, Ronayne Kate L, Towrie Michael
Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building, South Parks Road, Oxford OX1 3QU, United Kingdom.
J Phys Chem B. 2005 Aug 25;109(33):16099-108. doi: 10.1021/jp051315+.
Picosecond time-resolved mid-infrared absorption changes of the wild type green fluorescent protein from Aequorea victoria are reported on structural events during the photocycle. Concomitant with rapid H/D transfer following excitation of the neutral A state at 400 nm, a transient signal at 1721/1711 cm(-1) (H/D) developed belonging to protonated glutamate 222, which was definitively assigned using the E222D mutant from the altered proton-transfer kinetics to aspartate in addition to the altered band position and intensity in the spectra. A transient at 1697 cm(-1), assigned to a structural perturbation of glutamine 69, had a H/D kinetic isotope effect of >32, showing the conformational dynamics to be sensitive to the active site H/D vibrations. The kinetic data up to 2 ns after excitation in the 1250-1800 cm(-1) region in D2O provided 10 and 75 ps time constants for the excited-state deuteron transfer and the associated A1* - A1 and A2* - A2 difference spectra and showed the radiative intermediate I state vibrations and the transient accumulation of the long-lived ground-state intermediate I2. Assignments of chromophore modes for the A1, A2, and I2 ground states are proposed on the basis of published model compound studies (Esposito, A. P.; Schellenberg, P.; Parson, W. W.; Reid, P. J. J. Mol. Struct. 2001, 569, 25 and He, X.; Bell, A. F.; Tonge, P. J. J. Phys. Chem. B 2002, 106, 6056). Tentative assignments for the singlet-state intermediates A1*, A2*, and I* are discussed. An unexpected and unassigned band that may be a C=C chromophore vibration was observed in the ground state (1665 cm(-1)) as well as in all photocycle intermediates. Optical dumping of the transient I population was achieved using an additional 532 nm pulse and the directly obtained I2 - I* difference spectrum was highly similar to the I2 - I* photocycle spectrum. The pump-dump-probe spectrum differed from the pump-probe photocycle difference spectrum with respect to the intensity of the phenol 1 mode at 1578 cm(-1), suggesting stronger delocalization of the negative charge onto the phenolic ring of the anionic chromophore in the dumped I2 state. Indication for structural heterogeneity of the chromophore, Glu 222, and the chromophore environment was found in the two parallel proton-transfer reactions and their distinct associated ground- and intermediate-state vibrations. Vibrational spectral markers at 1695 cm(-1) assigned to Gln 69, at 1631 cm(-1) belonging to a C=C mode, and at 1354 cm(-1) belonging to a phenolate vibration further indicated the I2 and I* states to be unrelaxed.
报道了来自维多利亚多管水母的野生型绿色荧光蛋白在光循环过程中皮秒时间分辨的中红外吸收变化。在400nm激发中性A态后，伴随着快速的H/D转移，在1721/1711cm⁻¹（H/D）处出现了一个瞬态信号，该信号属于质子化的谷氨酸222，除了光谱中谱带位置和强度的改变外，利用E222D突变体中改变的质子转移动力学以及与天冬氨酸的差异，明确地将其归属。在1697cm⁻¹处的一个瞬态，归属为谷氨酰胺69的结构扰动，其H/D动力学同位素效应大于32，表明构象动力学对活性位点的H/D振动敏感。在D₂O中1250 - 1800cm⁻¹区域激发后2ns内的动力学数据，为激发态氘转移以及相关的A1* - A1和A2* - A2差光谱提供了10和75ps的时间常数，并显示了辐射中间I态振动和长寿命基态中间I2的瞬态积累。基于已发表的模型化合物研究（埃斯波西托，A.P.；谢伦贝格，P.；帕森，W.W.；里德，P.J.《分子结构杂志》2001年，569卷，25页以及何，X.；贝尔，A.F.；汤格，P.J.《物理化学杂志B》2002年，106卷，6056页），提出了A1、A2和I2基态发色团模式的归属。讨论了单重态中间体A1*、A2和I的初步归属。在基态（1665cm⁻¹）以及所有光循环中间体中都观察到了一个可能是C = C发色团振动的意外且未归属的谱带。使用额外的532nm脉冲实现了瞬态I群体的光猝灭，直接获得的I2 - I差光谱与I2 - I光循环光谱高度相似。泵浦 - 猝灭 - 探测光谱在1578cm⁻¹处酚1模式的强度方面与泵浦 - 探测光循环差光谱不同，这表明在猝灭的I2态中，负电荷在阴离子发色团的酚环上的离域更强。在两个平行的质子转移反应及其不同的相关基态和中间态振动中发现了发色团、谷氨酸222和发色团环境结构异质性的迹象。在1695cm⁻¹处归属为谷氨酰胺69、在1631cm⁻¹处属于C = C模式以及在1354cm⁻¹处属于酚盐振动的振动光谱标记进一步表明I2和I*态未弛豫。
