2-PHOTON SPECTROSCOPY OF LOCKED-11-CIS-RHODOPSIN - EVIDENCE FOR A PROTONATED SCHIFF-BASE IN A NEUTRAL PROTEIN-BINDING SITE
2-PHOTON SPECTROSCOPY OF LOCKED-11-CIS-RHODOPSIN - EVIDENCE FOR A PROTONATED SCHIFF-BASE IN A NEUTRAL PROTEIN-BINDING SITE
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DOI：
10.1073/pnas.82.12.4117
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
NAKANISHI, K
中科院分区：
文献类型：
--
作者：
BIRGE, RR;MURRAY, LP;NAKANISHI, K
用双光子光谱学研究了视紫红质的蛋白质结合部位的性质，以确定含有锁定的-11-顺式生色团的视紫红质模型中低位共价1AG*--类pi..pi.*状态的位置。在22,800 cm~(-1)，apprxeq处观察到2质子热透镜的极大值。2000 cm-1以上的1个光子吸收最大值，这表明蛋白质环境已经诱导了相对于在溶液中观察到的视黄基希夫碱的低位pi…pi*态的水平有序性反转。光谱结果清楚地表明，生色团是质子化的，结合位置没有变化。计算了结合位置的静电能量等高线图，指出了外部反离子的可能位置(S)。提出了两种结合位置的模型，以适应现有的光谱数据。一种模型涉及一个质子化的希夫碱发色团，它由一个带负电荷的天冬氨酸或谷氨酸残基稳定。还提出了一个更复杂的包含2个残基(一个带电荷，另一个中性)的模型。后一种模型很有趣，因为它还以质子在两个残基之间转移的形式考虑了观察到的氚同位素效应。移位被认为是一个基态过程，在生色团的光异构化之后开始，并通过异构化诱导的电荷分离导致的反离子环境的不稳定而能量驱动。
The nature of the protein binding site of rhodopsin was studied using 2-photon spectroscopy to assign the location of the low-lying covalent 1Ag*--like .pi..pi.* state in a model rhodopsin containing a locked-11-cis chromophore. The 2-proton thermal lens maximum is observed at 22,800 cm-1, .apprxeq. 2000 cm-1 above the 1 photon absorption maximum, indicating that the protein environment has induced a level ordering reversal of the low-lying .pi..pi.* states relative to that observed in retinyl Schiff bases in solution. The spectroscopic results clearly indicate that the chromophore is protonated and that the binding site is unchanged. Electrostatic energy contour maps of the binding site are calculated, showing possible locations for the external counterion(s). Two models of the binding site are proposed that accommodate the available spectroscopic data. One model involves a protonated Schiff base chromophore stabilized by a single negatively charged Asp or Glu residue. A more complicated model involving 2 residues (one charged, the other neutral) is also proposed. The latter model is interesting because it also accomodates the observed deuterium isotope effect in the form of a proton translocation between the 2 residues. The translocation is assumed to be a ground state process, initiated subsequent to the photoisomerization of the chromophore and energetically driven via destabilization of the counterion environment as a result of isomerization-induced charge separation.