Abstract
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 the Anabaena CTDH (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 obtain 1H, 13C and 15N resonance assignments. AnaCTDH samples enriched with 13C and 15N 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 with 15N 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.
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Abbreviations
- AnaCTDH:
-
C-terminal domain homolog of orange carotenoid protein from Anabaena variabilis
- NOE:
-
Nuclear Overhauser effect
- OCP:
-
Orange carotenoid protein
- τc :
-
Correlation time of protein tumbling
- R1 :
-
Longitudinal or spin–lattice relaxation rate
- R2 :
-
Transverse or spin–spin relaxation rate
- Rex :
-
Conformational exchange contribution to R2
- S2 :
-
Order parameter reflecting the amplitude of ps–ns bond vector dynamics
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Acknowledgements
The authors acknowledge the support of the Russian Science Foundation (Grant No. 18-44-04002) and the German Ministry for Education and Research (BMBF Grant No. 01DJ15007). Protein expression and purification were carried out in the frame of the Russian Foundation for Basic Research and the German Research Foundation joint Grant (No. 20-54-12018 and No. FR1276/5-1). The authors are grateful to Lomonosov Moscow State University (Russia) for the opportunity to use the NMR facilities. The authors acknowledge the support from the Russian Government Program of Competitive Growth of Kazan Federal University (KFU) among the world’s leading academic centers and the NMR equipment at the KFU Center of shared facilities. The 800 and 850 MHz NMR experiments were carried out using the equipment of the High-Field Nuclear Magnetic Resonance Center (HFNMRC) supported by Academia Sinica (AS-CFII-108-112), Taipei, Taiwan.
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Maksimov, E.G., Laptev, G.Y., Blokhin, D.S. et al. NMR resonance assignment and backbone dynamics of a C-terminal domain homolog of orange carotenoid protein. Biomol NMR Assign 15, 17–23 (2021). https://doi.org/10.1007/s12104-020-09976-1
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DOI: https://doi.org/10.1007/s12104-020-09976-1