The orange carotenoid protein (OCP) is a structurally and functionally modular photoactive protein involved in cyanobacterial photoprotection. Recently, based on bioinformatic analysis and phylogenetic relationships, new families of OCP have been described, OCP2 and OCPx. The first characterization of the OCP2 showed both faster photoconversion and back-conversion, and lower fluorescence quenching of phycobilisomes relative to the well-characterized OCP1. Moreover, OCP2 is not regulated by the fluorescence recovery protein (FRP). In this work, we present a comprehensive study combining ultrafast spectroscopy and structural analysis to compare the photoactivation mechanisms of OCP1 and OCP2 from Tolypothrix PCC 7601. We show that despite significant differences in their functional characteristics, the spectroscopic properties of OCP1 and OCP2 are comparable. This indicates that the OCP functionality is not directly related to the spectroscopic properties of the bound carotenoid. In addition, the structural analysis by X-ray footprinting reveals that, overall, OCP1 and OCP2 have grossly the same photoactivation mechanism. However, the OCP2 is less reactive to radiolytic labeling, suggesting that the protein is less flexible than OCP1. This observation could explain fast photoconversion of OCP2.
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.
- MeSH
- bakteriální proteiny chemie ultrastruktura MeSH
- kanthaxanthin chemie MeSH
- krystalografie rentgenová MeSH
- maloúhlový rozptyl MeSH
- nukleocytoplazmatické transportní proteiny chemie genetika ultrastruktura MeSH
- proteinové domény genetika MeSH
- sinice chemie ultrastruktura MeSH
- vazba proteinů účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The Helical Carotenoid Proteins (HCPs) are a large group of newly identified carotenoid-binding proteins found in ecophysiologically diverse cyanobacteria. They likely evolved before becoming the effector (quenching) domain of the modular Orange Carotenoid Protein (OCP). The number of discrete HCP families-at least nine-suggests they are involved in multiple distinct functions. Here we report the 1.7 Å crystal structure of HCP2, one of the most widespread HCPs found in nature, from the chromatically acclimating cyanobacterium Tolypothrix sp. PCC 7601. By purifying HCP2 from the native source we are able to identify its natively-bound carotenoid, which is exclusively canthaxanthin. In solution, HCP2 is a monomer with an absorbance maximum of 530 nm. However, the HCP2 crystals have a maximum absorbance at 548 nm, which is accounted by the stacking of the β1 rings of the carotenoid in the two molecules in the asymmetric unit. Our results demonstrate how HCPs provide a valuable system to study carotenoid-protein interactions and their spectroscopic implications, and contribute to efforts to understand the functional roles of this large, newly discovered family of pigment proteins, which to-date remain enigmatic.
- MeSH
- bakteriální proteiny chemie MeSH
- kanthaxanthin chemie MeSH
- krystalografie rentgenová MeSH
- proteinové domény MeSH
- sekundární struktura proteinů MeSH
- sinice chemie MeSH
- transportní proteiny chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The Orange Carotenoid Protein (OCP) is a photoactive water soluble protein that is crucial for photoprotection in cyanobacteria. When activated by blue-green light, it triggers quenching of phycobilisome fluorescence and regulates energy flow from the phycobilisome to the reaction center. The OCP contains a single pigment, the carotenoid 3'-hydroxyechinenone (hECN). Binding to the OCP causes a conformational change in hECN leading to an extension of its effective conjugation length. We have determined the S(1) energy of hECN in organic solvent and compared it with the S(1) energy of hECN bound to the OCP. In methanol and n-hexane, hECN has an S(1) energy of 14,300cm(-1), slightly higher than carotenoids with shorter conjugation lengths such as zeaxanthin or β-carotene; this is consistent with the proposal that the presence of the conjugated carbonyl group in hECN increases its S(1) energy. The S(1) energy of hECN in organic solvent is independent of solvent polarity. Upon binding to the OCP, the S(1) energy of hECN is further increased to 14,700cm(-1), underscoring the importance of protein binding which twists the conjugated carbonyl group into s-trans conformation and enhances the effect of the carbonyl group. Activated OCP, however, has an S(1) energy of 14,000cm(-1), indicating that significant changes in the vicinity of the conjugated carbonyl group occur upon activation.
We have studied spectroscopic properties of the 16kDa red carotenoid protein (RCP), which is closely related to the orange carotenoid protein (OCP) from cyanobacteria. Both proteins bind the same chromophore, the carotenoid 3'-hydroxyechinenone (hECN), and the major difference between the two proteins is lack of the C-terminal domain in the RCP; this results in exposure of part of the carotenoid. The excited-state lifetime of hECN in the RCP is 5.5ps, which is markedly longer than in OCP (3.3ps) but close to 6ps obtained for hECN in organic solvent. This confirms that the binding of hECN to the C-terminal domain in the OCP changes conformation of hECN, thereby altering its excited-state properties. Hydrogen bonds between the C-terminal domain and the carotenoid are also absent in the RCP. This allows the conformation of hECN in the RCP to be similar to that in solution, which results in comparable excited-state properties of hECN in solution. The red-shift of the RCP absorption spectrum is most likely due to aggregation of RCP induced by hydrophobic nature of hECN that, when exposed to buffer, stimulates formation of assemblies minimizing contact of hECN with water. We suggest that the loss of the C-terminal domain renders the protein amphipathic, containing both hydrophobic (the exposed part of hECN) and hydrophilic (N-terminal domain) regions, and may help the RCP to interact with lipid membranes; exposed hECN can penetrate into the hydrophobic environment of the lipid membrane, possibly to provide additional photoprotection.
- MeSH
- bakteriální proteiny chemie metabolismus MeSH
- cirkulární dichroismus MeSH
- karotenoidy chemie metabolismus MeSH
- konformace proteinů MeSH
- konzervovaná sekvence MeSH
- molekulární modely MeSH
- molekulová hmotnost MeSH
- sinice metabolismus MeSH
- spektrofotometrie MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
