Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography.
Volume-conserving trans-cis isomerization pathways in photoactive yellow protein visualized by picosecond X-ray crystallography.
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反式-顺式异构化是光活性蛋白质中的关键反应，通常不能通过标准的单键翻转机制发生。由于蛋白质环境施加的空间限制，异构化很可能通过“体积守恒”机制进行，在这种机制中，预计会有高度编排的原子运动，其细节尚未被直接观察到。在这里，我们使用时间分辨X射线结晶学对光活性黄蛋白中对香豆酸发色团的异构化进行了结构可视化，时间分辨率为100皮秒，空间分辨率为1.6°。最早的中间体(IT)的结构类似于高度应变的过渡态，其中扭转角位于反式和顺式异构体的中间。IT的反应轨迹分成两个结构截然不同的顺式中间体，分别为呼啦圈-扭转式和自行车-踏板式。通过E46Q突变减弱发色团和相邻残基之间的氢键，从而关闭自行车-踏板途径，可以控制分叉反应途径。
Trans-to-cis isomerization, the key reaction in photoactive proteins, cannot usually occur through the standard one-bond-flip mechanism. Due to spatial constraints imposed by a protein environment, isomerization is likely to proceed via a “volume-conserving” mechanism in which highly-choreographed atomic motions are expected, the details of which have not yet been directly observed. Here we employ time-resolved X-ray crystallography to structurally visualize isomerization of the p-coumaric acid chromophore in photoactive yellow protein with 100 picosecond time resolution and 1.6 Å spatial resolution. The structure of the earliest intermediate (IT) resembles a highly-strained transition state in which the torsion angle is located halfway between the trans and cis isomers. The reaction trajectory of IT bifurcates into two structurally distinct cis intermediates via hula-twist and bicycle-pedal pathways. The bifurcating reaction pathways can be controlled by weakening the hydrogen bond between the chromophore and an adjacent residue via E46Q mutation, which switches off the bicycle-pedal pathway.