NMR resonance assignment and backbone dynamics of a C-terminal domain homolog of orange carotenoid protein
NMR resonance assignment and backbone dynamics of a C-terminal domain homolog of orange carotenoid protein
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DOI：
10.1007/s12104-020-09976-1
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发表时间：
2020-09
影响因子：
0.9
通讯作者：
E. Maksimov;Gennady Yu Laptev;Dmitriy S. Blokhin;V. Klochkov;Y. B. Slonimskiy;N. Sluchanko;T. Friedrich;Chi-Fon Chang;V. Polshakov
中科院分区：
文献类型：
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作者：
E. Maksimov;Gennady Yu Laptev;Dmitriy S. Blokhin;V. Klochkov;Y. B. Slonimskiy;N. Sluchanko;T. Friedrich;Chi-Fon Chang;V. Polshakov
蓝藻中的光保护是由橙色类胡萝卜素蛋白（OCP）介导的，OCP是一种双结构域的光开关，具有其N端和c端多个天然同源结构域。最近，研究表明OCP的c -末端结构域同源物（CTDHs）是独立的类胡萝卜素蛋白，参与了膜和蛋白质之间的多向类胡萝卜素转移。类酮胡萝卜素的非共价包埋导致小的16 kda水溶性CTDH蛋白二聚化；然而，CTDH与膜和其他蛋白质的动态相互作用显然需要单体状态。虽然晶体学最近提供了anabaenactdh （AnaCTDH）在载子形式的空间结构的静态快照，预测了一些假定的功能段的迁移率，但目前还没有关于CTDH全息形式的晶体学信息。为了使用核磁共振技术来处理AnaCTDH蛋白的动力学，有必要获得1h，13C和15n的共振分配。利用重组蛋白表达制备了富含13c和15n同位素的AnaCTDH样品，并对90%以上的残基进行了核磁共振分配。结果表明，AnaCTDH在溶液中的结构与晶体中的结构基本相当。结合15n核磁共振弛豫实验，我们的数据揭示了AnaCTDH的动力学，为后续分析溶液中的全ctdh结构提供了平台，为更好地理解光触发的蛋白质-蛋白质相互作用和未来生物医学应用的抗氧化纳米载体的开发提供了平台。
Photoprotection in cyanobacteria is mediated by the Orange Carotenoid Protein (OCP), a two-domain photoswitch which has multiple natural homologs of its N- and C-terminal domains. Recently, it was demonstrated that C-terminal domain homologs (CTDHs) of OCP are standalone carotenoproteins participating in multidirectional carotenoid transfer between membranes and proteins. Non-covalent embedment of a ketocarotenoid causes dimerization of the small 16-kDa water-soluble CTDH protein; however, dynamic interactions of CTDH with membranes and other proteins apparently require the monomeric state. Although crystallography recently provided static snapshots of theAnabaenaCTDH (AnaCTDH) spatial structure in the apo-form, which predicted mobility of some putative functional segments, no crystallographic information on the holo-form of CTDH is presently available. In order to use NMR techniques to cope with the dynamics of the AnaCTDH protein, it was necessary to obtain1H,13C and15N resonance assignments. AnaCTDH samples enriched with13C and15N isotopes were prepared using recombinant protein expression, and NMR resonance assignment was achieved for more than 90% of the residues. The obtained results revealed that the structure of AnaCTDH in solution and in the crystal are largely equivalent. Together with15N NMR relaxation experiments, our data shed light on the AnaCTDH dynamics and provide the platform for the subsequent analysis of the holo-CTDH structure in solution, for the better understanding of light-triggered protein–protein interactions and the development of antioxidant nanocarriers for biomedical applications in the future.