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Dynamic structural changes of the photoactive orange carotenoid protein

Subject Area Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics
Biophysics
Structural Biology
Term from 2018 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 379950877
 
Final Report Year 2021

Final Report Abstract

The core objectives of the DynaStructOCP project were to obtain structural and spectroscopic data to (1) establish the sequence of light-induced transformations of the Orange Carotenoid Protein (OCP) at high time resolution, (2) to determine the stages of OCP’s interaction with its physiological regulator, the Fluorescence Recovery Protein (FRP) in molecular and structural detail. For this, low resolution structural methods in solution (small-angle X-ray/neutron scattering: SAXS, SANS), as well as high-resolution X-ray crystallography and NMR spectroscopy were applied. The structural dynamics was determined by femtosecond-resolved UV/Vis absorption, and picosecond-resolved fluorescence spectroscopy was carried out based on unprecedented pump-probe instrumentation. Regarding the universality of the FRP / OCP interaction, several low-homology species variants of FRP were tested with Synechocystis OCP, which revealed a correlation between the complex stoichiometry (2:1 and 1:1; detected by size exclusion chromatography / SAXS experiments) and the functional stage of the interaction. While 2:1 complexes were formed with photoactivated OCPR when attached to phycobilisomes, 1:1 complexes were beneficial for relaxation to the dark-adapted OCPO state. This was confirmed by the construction of FRP variants with redox-controllable mono- and dimerization properties, since mandatory FRP dimers were required for terminating OCPR interaction with phycobilisomes, while the ability to monomerize facilitated the OCPR→OCPO relaxation. Comparative SAXS and SANS studies of wild-type OCP and an OCPR state analogue showed that both species are dimeric in solution, but the OCPR state exhibits a strongly elongated shape with substantially increased flexibility due to domain separation and unfolding of connecting loops. The binding site for FRP on the β-sheet surface of OCP’s C-terminal domain was determined, and the dimer interface between two FRP molecules could be excluded for the interaction with OCP. QENS studies revealed substantially larger protein and surface water dynamics of the OCPR state analogue, both of which could be a prerequisite for efficient physiological energy dissipation by the OCPR form. Ultrafast spectroscopy benefitted from construction of OCP variants with superior spectral properties. In the OCP-3FH construct, all but the most important Trp residue (Trp-288) were replaced by Phe or His residues allowing us to monitor the implication of Trp-288 in H-bonding to the echinenone (ECN) cofactor by fluorescence spectroscopy at picosecond resolution. After an actinic flash, Trp-288 fluorescence increased within 23 ps, which correlates with the time course of the S* intermediate, a distinct spectral feature on the blue flange (550-600 nm) of the S1-SN absorption, which is considered to be the key for the breaking of conserved H-bonds as a prerequisite for the productive photocycle. Subsequent transient changes of Trp-288 fluorescence on the time scale of milliseconds correlated with the first conformational rearrangements of the protein matrix before domain separation occurs. Ultrafast absorption experiments employed the OCP-Y201W variant, which displayed unprecedented purity of the OCPO ground state, as rationalized based on determination of the X-ray crystal structure (PDB codes 6T6K, 6T6M, 6T6O). This structure revealed that the Trp in position 201 can assume two orientations, both of which weakening or removing one H-bond to ECN and augmenting the importance of the remaining H-bond. Femtosecond-resolved UV/Vis spectroscopy revealed 25 % yield of the S* photoproduct, which occurs in parallel to H-bond disruption between Trp-288 and the ECN cofactor. Based on the crystal structure and quantum chemical calculations, we suggested a mechanism, in which the formation of an oxocarbenium ion on the ketocarotenoid may precede and facilitate the release of the carotenoid for the ultrafast formation of red-absorbing states of OCP in the early photocycle. Structural research resulted in the solution of the X-ray crystal structure (PDB code 6FEJ) of the Anabaena homolog of OCP’s C-terminal domain as a model for the C-terminal domain in the OCPR state. This study revealed the importance of the β5-β6 loop and the C-terminal tail on the ability of the protein to shuttle carotenoid molecules between membranes and/or proteins. NMR experiments with this protein led to chemical shift assignments of 89% of the backbone residues, which together with relaxation data were deposited in the BMRB database (acc. no. BMRB-50410). Finally, a concept for a fluorescent temperature sensor was developed based on fusions between fluorescent proteins and OCP, which permits temperature determination with 0.1 °C accuracy.

Publications

  • (2018). Functional interaction of low-homology FRPs from different cyanobacteria with Synechocystis OCP. Biochim Biophys Acta Bioenerg. 1859(5):382- 393
    Slonimskiy, Y.B., Maksimov, E.G., Lukashev, E.P., Moldenhauer, M., Jeffries, C.M., Svergun, D.I., Friedrich, T., Sluchanko, N.N.
    (See online at https://doi.org/10.1016/j.bbabio.2018.03.001)
  • (2018). OCP-FRP protein complex topologies suggest a mechanism for controlling high light tolerance in cyanobacteria. Nat Commun. 9(1):3869
    Sluchanko, N.N., Slonimskiy, Y.B., Shirshin, E.A., Moldenhauer, M., Friedrich, T., Maksimov, E.G
    (See online at https://doi.org/10.1038/s41467-018-06195-0)
  • (2018). Structural rearrangements in the C-terminal domain homolog of Orange Carotenoid Protein are crucial for carotenoid transfer. Commun Biol. 1:125
    Harris, D., Wilson, A., Muzzopappa, F., Sluchanko, N.N., Friedrich, T., Maksimov, E.G., Kirilovsky, D., Adir, N.
    (See online at https://doi.org/10.1038/s42003-018-0132-5)
  • (2019). Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein. Part II: Quasielastic Neutron Scattering. J Phys Chem B. 123(45):9536-9545
    Golub, M., Moldenhauer, M., Schmitt, F.-J., Lohstroh, W., Maksimov, E.G., Friedrich, T., Pieper, J.
    (See online at https://doi.org/10.1021/acs.jpcb.9b05073)
  • (2019). Solution Structure and Conformational Flexibility in the Active State of the Orange Carotenoid Protein: Part I. Small-Angle Scattering. J Phys Chem B. 123(45):9525-9535
    Golub, M., Moldenhauer, M., Schmitt, F.-J., Feoktystov, A., Mändar, H., Maksimov, E., Friedrich, T., Pieper, J.
    (See online at https://doi.org/10.1021/acs.jpcb.9b05071)
  • (2019). Structural peculiarities of ketocarotenoids in water-soluble proteins revealed by simulation of linear absorption. Phys Chem Chem Phys. 21(46):25707-25719
    Pishchalnikov, R.Y., Yaroshevich, I.A., Slastnikova, T.A., Ashikhmin, A.A., Stepanov, A.V., Slutskaya, E.A., Friedrich, T., Sluchanko, N.N., Maksimov, E.G.
    (See online at https://doi.org/10.1039/c9cp04508b)
  • (2020). Engineering the photoactive orange carotenoid protein with redoxcontrollable structural dynamics and photoprotective function. Biochim Biophys Acta Bioenerg. 1861(5-6):148174
    Slonimskiy, Y.B., Maksimov, E.G., Lukashev, E.P., Moldenhauer, M., Friedrich, T., Sluchanko, N.N.
    (See online at https://doi.org/10.1016/j.bbabio.2020.148174)
  • (2020). Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein. Sci Rep. 10(1):11729
    Maksimov, E.G., Protasova, E.A., Tsoraev, G.V., Yaroshevich, I.A., Maydykovskiy, A.I., Shirshin, E.A., Gostev, T.S., Jelzow, A., Moldenhauer, M., Slonimskiy, Y.B., Sluchanko, N.N., Friedrich, T.
    (See online at https://doi.org/10.1038/s41598-020-68463-8)
  • (2021). NMR resonance assignment and backbone dynamics of a C-terminal domain homolog of orange carotenoid protein. Biomol NMR Assign. 15, 17–23
    Maksimov, E.G., Laptev, G.Y., Blokhin, D.S., Klochkov, V.V., Slonimskiy, Y.B., Sluchanko, N.N., Friedrich, T., Chang, C.F., Polshakov, V.I.
    (See online at https://doi.org/10.1007/s12104-020-09976-1)
  • (2021). Role of hydrogen bond alternation and charge transfer states in photoactivation of the Orange Carotenoid Protein. Comms. Biol. 4:539
    Yaroshevich, I.A., Maksimov, E.G., Sluchanko, N.N., Zlenko, D.V., Stepanov, A.V., Slutskaya, E.A., Slonimskiy, Y.B., Botnarevskii, V.S., Remeeva, A., Gushchin, I., Kovalev, K., Gordeliy, V.I., Shelaev, I.V., Gostev, F.E., Khakhulin, D., Poddubnyy, V.V., Gostev, T.S., Cherepanov, D.A., Polívka, T., Kloz, M., Friedrich, T., Paschenko, V.Z., Nadtochenko, V.A., Rubin, A.B., Kirpichnikov, M.P.
    (See online at https://doi.org/10.1038/s42003-021-02022-3)
 
 

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