Structural and spectroscopic characterization of HCP2

Domínguez-Martín, María Agustina; Polı́vka, Tomáš; Sutter, Markus; Ferlez, Bryan; Lechno‐Yossef, Sigal; Montgomery, Beronda L.; Kerfeld, Cheryl A.

doi:10.1016/j.bbabio.2019.03.004

Cited by 22 publications

(73 citation statements)

References 56 publications

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“…For comparison, the largest known carotenoid shift (> 100 nm) is the binding of asthaxanthin to the lobster protein crustacyanin 5 . Some OCP mutants exhibit absorption maximum at around 525 nm 41 , and HCP binding CAN has a peak absorption at ~ 530 nm 9,14 . The origin of this significant red-shift is most likely due to CCP-specific protein-carotenoid interactions; Y28 and W111 likely hydrogen bond to the keto-oxygen of the CAN in CCP2.…”

Section: Discussionmentioning

confidence: 99%

“…The CCP2 overexpression strain was grown in buffered BG-11 pH 8.0 medium supplemented with 25 μg/mL of kanamycin. The holo-CCP2 was isolated according to the method described previously 14 with a modified lysis buffer (50 mM Tris pH 8.0, 200 mM NaCl, and 10% glycerol containing DNase I ( Sigma-Aldrich ), protease inhibitor cocktail ( Sigma-Aldrich ), Lysozyme ( Sigma-Aldrich ), 1 mM EDTA and 0.5 mM PMSF). 30 mg of purified CCP2 was obtained from an 18 L culture.…”

Section: Methodsmentioning

confidence: 99%

“…To produce apo-CCP2 in E. coli , C-terminally His-tagged (6X-His) Tolypothrix CCP2 was generated by PCR using primers SL25 (5′-TCCTTAGTGATGATGGTGGTGATGATCACGACGCATAGCGA-3′) and SL26 (5′-CCATGGGCACTACTGCTGAATTTAACAA-3′), adding a His-Tag on the reverse primer, cloned in pET16b vector ( Novagen ) between NcoI and BamHI sites and expressed in BL21(DE3) cells. The purification of apo-CCP2 was carried out as previously described 14 .…”

Section: Methodsmentioning

confidence: 99%

“…This procedure was repeated several times until a white pellet was obtained, indicating the absence of pigment in the protein sample. The carotenoid composition was analyzed as described previously using LC-DAD-MS 14 .…”

Section: Methodsmentioning

confidence: 99%

“…Because HCPs and CCPs are water-soluble carotenoproteins they are valuable model systems to probe carotenoid-protein interactions and their spectral manifestations. Recently, HCPs have begun to be biochemically and structurally characterized 9 , 13 – 15 , revealing that the HCPs are monomers that bind a single carotenoid 9 , 14 .…”

Section: Introductionmentioning

confidence: 99%

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Structural analysis of a new carotenoid-binding protein: the C-terminal domain homolog of the OCP

Domínguez-Martín

Hammel

Gupta

et al. 2020

Sci Rep

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The Orange Carotenoid Protein (OCP) is a water-soluble protein that governs photoprotection in many cyanobacteria. The 35 kDa OCP is structurally and functionally modular, consisting of an N-terminal effector domain (NTD) and a C-terminal regulatory domain (CTD); a carotenoid spans the two domains. The CTD is a member of the ubiquitous Nuclear Transport Factor-2 (NTF2) superfamily (pfam02136). With the increasing availability of cyanobacterial genomes, bioinformatic analysis has revealed the existence of a new family of proteins, homologs to the CTD, the C-terminal domain-like carotenoid proteins (CCPs). Here we purify holo-CCP2 directly from cyanobacteria and establish that it natively binds canthaxanthin (CAN). We use small-angle X-ray scattering (SAXS) to characterize the structure of this carotenoprotein in two distinct oligomeric states. A single carotenoid molecule spans the two CCPs in the dimer. Our analysis with X-ray footprinting-mass spectrometry (XFMS) identifies critical residues for carotenoid binding that likely contribute to the extreme red shift (ca. 80 nm) of the absorption maximum of the carotenoid bound by the CCP2 dimer and a further 10 nm shift in the tetramer form. These data provide the first structural description of carotenoid binding by a protein consisting of only an NTF2 domain.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Structural analysis of a new carotenoid-binding protein: the C-terminal domain homolog of the OCP

Domínguez-Martín

Hammel

Gupta

et al. 2020

Sci Rep

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Excited‐State Evolution of Keto‐Carotenoids after Excess Energy Excitation in the UV Region

et al. 2021

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Carotenoids are molecules with rich photophysics that are in many biological systems involved in photoprotection. Yet, their response to excess energy excitation is only scarcely studied. Here we have explored excited state properties of three keto‐carotenoids, echinenone, canthaxanthin and rhodoxanthin after excess energy excitation to a singlet state absorbing in UV. Though the basic spectral features and kinetics of S2, hot S1, relaxed S1 states remain unchanged upon UV excitation, the clear increase of the S* signal is observed after excess energy excitation, associated with increased S* lifetime. A multiple origin of the S* signal, originating either from specific conformations in the S1 state or from a non‐equilibrated ground state, is confirmed in this work. We propose that the increased amount of energy stored in molecular vibrations, induced by the UV excitation, is the reason for the enhanced S* signal observed after UV excitation. Our data also suggest that a fraction of the UV excited state population may proceed through a non‐sequential pathway, bypassing the S2 state.

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UV Excitation of Carotenoid Binding Proteins OCP and HCP: Excited‐State Dynamics and Product Formation

et al. 2021

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Over the last decade the orange carotenoid protein (OCP) gained attention due to its role in photoprotection in cyanobacteria. Recently, other carotenoid binding proteins such as helical carotenoid proteins (HCPs) have been identified and characterized, although the function of the HCPs is not yet clarified. Here we have examined OCP1 and two HCPs, HCP2 and HCP3, from Tolypothrix PCC 7601 by ultrafast spectroscopy after excitation of both carotenoid canthaxanthin and amino acids tryptophan and tyrosine in UV at 275 nm. We find that the canthaxanthin S 1 lifetime remains the same 3.5 ps as for excitation of the carotenoid S 2 state, but the S* signal is significantly amplified by UV excitation. In contrast to the S 2 excitation, we observe a formation of the canthaxanthin radical cation at 900 nm for all three proteins after 275 nm excitation. In OCP1, UV light also enhances the amplitude of spectral band centered at 550 nm at longer delays. This band likely corresponds to the first photoproduct of the OCP photocycle, suggesting that excess energy excitation and/or direct excitation of amino acids can enhance the product formation.

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Structural and spectroscopic characterization of HCP2

Cited by 22 publications

References 56 publications

Structural analysis of a new carotenoid-binding protein: the C-terminal domain homolog of the OCP

Structural analysis of a new carotenoid-binding protein: the C-terminal domain homolog of the OCP

Excited‐State Evolution of Keto‐Carotenoids after Excess Energy Excitation in the UV Region

UV Excitation of Carotenoid Binding Proteins OCP and HCP: Excited‐State Dynamics and Product Formation

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