Department of Physics, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya, Japan.
Proteins. 2012 Oct;80(10):2384-96. doi: 10.1002/prot.24124. Epub 2012 Jun 30.
The lifetime of the O intermediate of bacteriorhodopsin (BR) is extended by a factor of ∼250 in the Leu93-to-Ala mutant (BR_L93A). To clarify the structural changes occurring in the last stage of the proton pumping cycle of BR, we crystallized BR_L93A into a hexagonal P622 crystal. Diffraction data from the unphotolyzed state showed that the deletion of three carbon atoms from Leu93 is compensated by the insertion of four water molecules in the cytoplasmic vicinity of retinal. This insertion of water is suggested to be responsible for the blue-shifted λ(max) (540 nm) of the mutant. A long-lived substate of O with a red-shifted λ(max) (~565 nm) was trapped when the crystal of BR_L93A was flash-cooled after illumination with green light. This substate (O(slow)) bears considerable similarity to the M intermediate of native BR; that is, it commonly shows deformation of helix C and the FG loop, downward orientation of the side chain of Arg82, and disruption of the Glu194/Glu204 pair. In O(slow), however, the main chain of Lys216 is less distorted and retinal takes on the 13-cis/15-syn configuration. Another significant difference is seen in the pH dependence of the structure of the proton release group, the pK(a) value of which is suggested to be much lower in O(slow) than in M.
紫膜质菌视紫红质（BR）O 中间态的寿命在 Leu93 到 Ala 突变体（BR_L93A）中延长了约 250 倍。为了阐明 BR 质子泵循环最后阶段发生的结构变化，我们将 BR_L93A 结晶成六方 P622 晶体。来自未光解状态的衍射数据表明，Leu93 缺失三个碳原子被四个水分子插入到视黄醛的细胞质附近所补偿。这种水分子的插入被认为是导致突变体 λ(max)（540nm）蓝移的原因。当 BR_L93A 的晶体在绿光照射后快速冷却时，捕获到具有红移 λ(max)（~565nm）的 O 长寿命亚基（O(slow)）。这个亚基（O(slow)）与天然 BR 的 M 中间态非常相似；也就是说，它通常表现出 C 螺旋和 FG 环的变形、Arg82 侧链的向下取向以及 Glu194/Glu204 对的破坏。然而，在 O(slow)中，Lys216 的主链扭曲程度较小，并且视黄醛采用 13-顺式/15-顺式构型。另一个显著的区别是在质子释放基团结构的 pH 依赖性方面，O(slow)中质子释放基团的 pK(a)值被认为比 M 中低得多。
