直接观察离子泵中离子运输过程中蛋白质结构的变化为离子运输机制提供了宝贵的见解。在这项研究中,我们检查了亚毫秒时间尺度上的光驱动钠离子(Na)泵视紫红质KR2的结构变化,与Na+的吸收和释放相对应。我们比较了WT和W215F突变体的离子泵浦活性和瞬态吸收光谱,其中位于细胞质侧的视网膜发色团附近的Trp215残基被Phe残基取代。我们的发现表明,Trp215的庞大侧链与视网膜发色团的C20甲基之间的原子接触可促进视网膜发色团从13顺式到全反式形式的松弛。由于Trp215在其他离子泵浦视紫红质中是保守的,目前的结果表明,这种残留物通常充当机械传感器。此外,我们测量了时间分辨的紫外共振拉曼(UVRR)光谱,以表明Trp215周围的环境在光辐射后1ms变得疏水性较低,并恢复到未光解状态,时间常数约为10ms。这些时间尺度对应于Na+的摄取和释放,表明Na+摄取的细胞质侧的瞬时离子通道的演化,与离子泵的交替接入模型一致。时间分辨UVRR技术具有应用于其他离子泵浦视紫红质的潜力,并且可以提供对离子传输机制的进一步见解。
Direct observation of protein structural changes during ion transport in ion pumps provides valuable insights into the mechanism of ion transport. In this study, we examined structural changes in the light-driven sodium ion (Na+) pump rhodopsin KR2 on the sub-millisecond time scale, corresponding with the uptake and release of Na+. We compared the ion-pumping activities and transient absorption spectra of WT and the W215F mutant, in which the Trp215 residue located near the retinal chromophore on the cytoplasmic side was replaced with a Phe residue. Our findings indicated that atomic contacts between the bulky side chain of Trp215 and the C20 methyl group of the retinal chromophore promote relaxation of the retinal chromophore from the 13-cis to the all-trans form. Since Trp215 is conserved in other ion-pumping rhodopsins, the present results suggest that this residue commonly acts as a mechanical transducer. In addition, we measured time-resolved ultraviolet resonance Raman (UVRR) spectra to show that the environment around Trp215 becomes less hydrophobic at 1 ms after photoirradiation and recovers to the unphotolyzed state with a time constant of around 10 ms. These time scales correspond to Na+ uptake and release, suggesting evolution of a transient ion channel at the cytoplasmic side for Na+ uptake, consistent with the alternating-access model of ion pumps. The time-resolved UVRR technique has potential for application to other ion-pumping rhodopsins and could provide further insights into the mechanism of ion transport.