Kim Tae Wu, Yang Cheolhee, Kim Youngmin, Kim Jong Goo, Kim Jeongho, Jung Yang Ouk, Jun Sunhong, Lee Sang Jin, Park Sungjun, Kosheleva Irina, Henning Robert, van Thor Jasper J, Ihee Hyotcherl
Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, 305-701, Korea.
Center for Nanomaterials and Chemical Reactions, Institute for Basic Science (IBS), Daejeon, 305-701, Korea.
Phys Chem Chem Phys. 2016 Apr 7;18(13):8911-8919. doi: 10.1039/c6cp00476h.
Real-time probing of structural transitions of a photoactive protein is challenging owing to the lack of a universal time-resolved technique that can probe the changes in both global conformation and light-absorbing chromophores of the protein. In this work, we combine time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) spectroscopy to investigate how the global conformational changes involved in the photoinduced signal transduction of photoactive yellow protein (PYP) is temporally and spatially related to the local structural change around the light-absorbing chromophore. In particular, we examine the role of internal proton transfer in developing a signaling state of PYP by employing its E46Q mutant (E46Q-PYP), where the internal proton transfer is inhibited by the replacement of a proton donor. The comparison of TRXSS and TA spectroscopy data directly reveals that the global conformational change of the protein, which is probed by TRXSS, is temporally delayed by tens of microseconds from the local structural change of the chromophore, which is probed by TA spectroscopy. The molecular shape of the signaling state reconstructed from the TRXSS curves directly visualizes the three-dimensional conformations of protein intermediates and reveals that the smaller structural change in E46Q-PYP than in wild-type PYP suggested by previous studies is manifested in terms of much smaller protrusion, confirming that the signaling state of E46Q-PYP is only partially developed compared with that of wild-type PYP. This finding provides direct evidence of how the environmental change in the vicinity of the chromophore alters the conformational change of the entire protein matrix.
由于缺乏一种能同时探测蛋白质整体构象变化和吸光发色团变化的通用时间分辨技术，对光活性蛋白的结构转变进行实时探测具有挑战性。在这项工作中，我们结合时间分辨X射线溶液散射（TRXSS）和瞬态吸收（TA）光谱，来研究光活性黄色蛋白（PYP）光诱导信号转导过程中涉及的整体构象变化如何在时间和空间上与吸光发色团周围的局部结构变化相关。特别地，我们通过使用其E46Q突变体（E46Q - PYP）来研究内部质子转移在PYP信号状态形成中的作用，在该突变体中，质子供体的替换抑制了内部质子转移。TRXSS和TA光谱数据的比较直接表明，由TRXSS探测到的蛋白质整体构象变化在时间上比由TA光谱探测到的发色团局部结构变化延迟了数十微秒。从TRXSS曲线重建的信号状态的分子形状直接可视化了蛋白质中间体的三维构象，并揭示了先前研究表明的E46Q - PYP中比野生型PYP更小的结构变化表现为更小的突出，证实与野生型PYP相比，E46Q - PYP的信号状态仅部分形成。这一发现为发色团附近的环境变化如何改变整个蛋白质基质的构象变化提供了直接证据。
