Milliseconds to seconds timescale, filling the gap in the photo-reaction of carH with time-resolved X-ray scattering in solution.

The protein carH associates with the B12 vitamin to form a light-sensitive gene repressor in bacteria. The gene repressive state in the dark binds to the DNA as a tetramer. The light triggers the dissociation of the adenosyl group from the cobalamin, the coordination of the cobalt to the H132 of carH and the dissociation of the tetramer. We recently obtained time-resolved wide-angle X-ray scattering (TR-WAXS) results regarding the tetramer to monomer transition of a truncated form of carH (TtCBD) but we still lack information about the oligomerization change for the full-length carH and for two other variants, the TtH132A mutant and the HmCBD homologue. To this end, TR-WAXS will be used on ID09. Preliminary results on TtCBD show the presence of two structural intermediates relaxing within 3 ms and 300 ms before the tetramer dissociation occurs. We aim at collecting data on the other carH variants for long time delays ranging from 100 ms to 5 s as recently achieved on ID09 for TtCBD.

carH蛋白可与维生素B12结合，在细菌中组装为光敏基因阻遏复合物。该复合物在黑暗条件下以四聚体形式结合DNA，实现基因转录阻遏。光照可触发钴胺素上腺苷基团的解离、钴离子与carH蛋白H132位点的配位结合，以及四聚体的解离。我们此前已针对截短型carH（TtCBD）的四聚体-单体转变过程，获取了时间分辨广角X射线散射（time-resolved wide-angle X-ray scattering, TR-WAXS）的实验数据，但目前仍缺乏全长carH，以及另外两种变体——TtH132A突变体与HmCBD同源蛋白——的寡聚化状态变化相关信息。为此，本研究将在同步辐射ID09线站上开展TR-WAXS实验。针对TtCBD的预实验结果显示，在四聚体解离发生前，存在两种结构中间体，其弛豫过程分别在3 ms与300 ms内完成。我们计划针对其余carH变体，采集延迟时间范围为100 ms至5 s的实验数据，该参数范围已在此前ID09线站针对TtCBD的实验中实现。