Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein
Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein
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DOI：
10.1038/s41598-020-68463-8
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发表时间：
2020-07-16
影响因子：
4.6
通讯作者：
Friedrich, Thomas
中科院分区：
文献类型：
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作者：
Maksimov, Eugene G.;Protasova, Elena A.;Friedrich, Thomas
光活性橙类胡萝卜素蛋白（OCP）在蓝藻的光保护中起关键作用。在OCP中，单个非共价结合的类酮胡萝卜素分子充当光强度传感器，而蛋白质负责与光收集天线形成分子接触，其荧光被OCP猝灭。这种生理相互作用的激活需要从光激发的类胡萝卜素到蛋白质基质的信号转导。最近的研究揭示了类胡萝卜素和蛋白质之间的构象转变是不同步的。内在色氨酸（Trp）荧光为OCP光循环过程中的蛋白质部分提供了有价值的信息。然而，野生型OCP含有5个Trp残基，这使得提取位点特异性信息变得不可能。在这项工作中，我们克服了这一问题，通过表征全光活性OCP变体（OCP- 3fh）的光循环，只有最关键的色氨酸残基（色氨酸288）在适当的位置。Trp-288是特别有趣的，因为它与类胡萝卜素的酮氧形成氢键，以保持OCP处于黑暗适应状态。利用飞秒泵探针荧光光谱分析了OCP- 3fh的光循环，并确定了第一个中间体的形成速率，这表明最近发现的类胡萝卜素在OCP中S*态的产生早于氢键的断裂。因此，根据独特的光活性OCP- 3fh变体的色氨酸荧光，我们确定了氢键断裂的速率，并为野生型OCP伴随光激活的早期事件提供了新的见解。
The photoactive Orange Carotenoid Protein (OCP) plays a key role in cyanobacterial photoprotection. In OCP, a single non-covalently bound keto-carotenoid molecule acts as a light intensity sensor, while the protein is responsible for forming molecular contacts with the light-harvesting antenna, the fluorescence of which is quenched by OCP. Activation of this physiological interaction requires signal transduction from the photoexcited carotenoid to the protein matrix. Recent works revealed an asynchrony between conformational transitions of the carotenoid and the protein. Intrinsic tryptophan (Trp) fluorescence has provided valuable information about the protein part of OCP during its photocycle. However, wild-type OCP contains five Trp residues, which makes extraction of site-specific information impossible. In this work, we overcame this problem by characterizing the photocycle of a fully photoactive OCP variant (OCP-3FH) with only the most critical tryptophan residue (Trp-288) in place. Trp-288 is of special interest because it forms a hydrogen bond to the carotenoid's keto-oxygen to keep OCP in its dark-adapted state. Using femtosecond pump-probe fluorescence spectroscopy we analyzed the photocycle of OCP-3FH and determined the formation rate of the very first intermediate suggesting that generation of the recently discovered S* state of the carotenoid in OCP precedes the breakage of the hydrogen bonds. Therefore, following Trp fluorescence of the unique photoactive OCP-3FH variant, we identified the rate of the H-bond breakage and provided novel insights into early events accompanying photoactivation of wild-type OCP.