Tittor J, Paula S, Subramaniam S, Heberle J, Henderson R, Oesterhelt D
Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany.
J Mol Biol. 2002 May 31;319(2):555-65. doi: 10.1016/S0022-2836(02)00307-8.
Unlike wild-type bacteriorhodopsin (BR), the BR triple mutant D96G/F171C/F219L has been shown to undergo only minor structural rearrangements during its photocycle. Nonetheless, the mutant is capable of transporting protons at a rate of 125(+/-40) H+/BR per minute under light-saturating conditions. Light adaptation of the triple mutant's retinal proceeds in a pH-dependent manner up to a maximum of 63% all-trans. These two findings imply that the transport activity of the triple mutant comprises 66% of the wild-type activity. Time-resolved spectroscopy reveals that the identity and sequence of intermediates in the photocycle of the triple mutant in the all-trans configuration correspond to that of wild-type BR. The only differences relate to a slower rise and decay of the M and O intermediates, and a significant spectral contribution from a 13-cis component. No indication for accumulation of the N intermediate is found under a variety of conditions that normally favor the formation of this species in wild-type BR. The Fourier transform infrared (FTIR) spectrum of the M intermediate in the triple mutant resembles that of wild type. Minor changes in the amide I region during the photocycle suggest that only small movements of the protein backbone occur. Electron microscopy reveals large differences in conformation between the unilluminated state of the mutant protein and wild-type but no light-induced changes in time-resolved measurements. Evidently, proton transport by the triple mutant does not require the major conformational rearrangements that occur on the same time-scale with wild-type. Thus, we conclude that large conformational changes observed in the photocycle of the wild-type and many BR mutants are not a prerequisite for the change in accessibility of the Schiff base nitrogen atom that must occur during vectorial catalysis to allow proton transport.
与野生型细菌视紫红质（BR）不同，BR三重突变体D96G/F171C/F219L在其光循环过程中仅发生微小的结构重排。尽管如此，该突变体在光饱和条件下能够以每分钟125（±40）个H⁺/BR的速率转运质子。三重突变体视网膜的光适应以pH依赖的方式进行，最高可达63%的全反式。这两个发现表明三重突变体的转运活性占野生型活性的66%。时间分辨光谱显示，全反式构型的三重突变体光循环中间体的特性和序列与野生型BR的一致。唯一的差异在于M和O中间体的上升和衰减较慢，以及13-顺式组分有显著的光谱贡献。在通常有利于野生型BR形成该物种的各种条件下，未发现N中间体积累的迹象。三重突变体中M中间体的傅里叶变换红外（FTIR）光谱与野生型相似。光循环过程中酰胺I区域的微小变化表明蛋白质主链仅发生小的移动。电子显微镜显示突变蛋白的未光照状态与野生型在构象上有很大差异，但在时间分辨测量中未发现光诱导变化。显然，三重突变体的质子转运不需要与野生型在同一时间尺度上发生的主要构象重排。因此，我们得出结论，在野生型和许多BR突变体的光循环中观察到的大的构象变化不是矢量催化过程中席夫碱氮原子可及性变化的先决条件，而矢量催化过程中席夫碱氮原子可及性变化是质子转运所必需的。
