The Unique Protein-to-Protein Carotenoid Transfer Mechanism

Abstract

Orange Carotenoid Protein (OCP) is known as an effector and regulator of cyanobacterial photoprotection. This 35 kDa water-soluble protein provides specific environment for blue-green light absorbing keto-carotenoids, which excitation causes dramatic but fully reversible rearrangements of the OCP structure, including carotenoid translocation and separation of C- and N-terminal domains upon transition from the basic orange to photoactivated red OCP form. Although recent studies greatly improved our understanding of the OCP photocycle and interaction with phycobilisomes and the fluorescence recovery protein, the mechanism of OCP assembly remains unclear. Apparently, this process requires targeted delivery and incorporation of a highly hydrophobic carotenoid molecule into the water-soluble apoprotein of OCP. Recently, we introduced, to our knowledge, a novel carotenoid carrier protein, COCP, which consists of dimerized C-domain(s) of OCP and can combine with the isolated N-domain to form transient OCP-like species. Here, we demonstrate that in vitro COCP efficiently transfers otherwise tightly bound carotenoid to the full-length OCP apoprotein, resulting in formation of photoactive OCP from completely photoinactive species. We accurately analyze the peculiarities of this process that, to the best of our knowledge, appears unique, a previously uncharacterized protein-to-protein carotenoid transfer mechanism. We hypothesize that a similar OCP assembly can occur in vivo, substantiating specific roles of the COCP carotenoid carrier in cyanobacterial photoprotection.

Figure 1

Carotenoid transfer from COCP to Apo-OCP followed by absorption spectroscopy. (A) Absorption spectrum and color of the carotenoid bound to COCP undergo significant changes upon addition of Apo-OCP (3 Apo-OCP per 1 COCP dimer), which gradually turns the sample from violet into red and, finally, into orange. The spectrum of species obtained after mixing of COCP and an excess of Apo-OCP (line 1) represents a mixture of two forms—orange (dashed line), reminiscent of the OCP^O spectrum, and violet (dotted line), similar to the absorption spectrum of initial COCP solution. (B) Characteristic time-course of O.D. at 550 nm was measured upon addition of Apo-OCP to the solution of carotenoid-containing COCP (V, violet) resulting in formation of a typical orange-like OCP form (O), which is photoactive and could be converted into the red form (R). (C) Shown here are changes of O.D. at 100 s after addition of different Apo-OCP concentrations to the solution of COCP dimer at 33°C. (D) Shown here are time-courses of carotenoid transfer upon addition of Apo-OCP to COCP (1), Apo-OCP to COCP-W288A (2), and Apo-ΔNTE-OCP to COCP (3). (E) Given here are Arrhenius plots of the rates of the orange form for COCP and COCP-W288A as the initial sources of carotenoid. Dependences were approximated by linear functions to estimate the activation energies. To see this figure in color, go online.

Figure 2

Oligomeric status of COCP-W288A, OCP^R, and Apo-OCP proteins studied by analytical SEC on a Superdex 200 Increase column (GE Healthcare Life Sciences). (A) Given here are SEC profiles of COCP-W288A (inset) obtained at different protein concentrations loaded on a column and followed by carotenoid-specific absorbance. (B) Given here are dependencies of the apparent MW of different OCP-related species on protein concentration in the loaded sample. (C) Given here are SEC profiles of the OCP^WT sample preilluminated on ice and loaded at different protein concentrations on a constantly blue-LED illuminated SEC column. (D) Given here are SEC profiles for different protein concentrations of Apo-OCP followed by protein-specific absorbance. Flow rate was 1.2 mL/min, temperature was 23°C. The results were reproduced at least two times for each case. To see this figure in color, go online.

Figure 3

Analysis of Apo-OCP and OCP^O by SAXS. (A) SEC profiles of Apo-OCP (200 μM) and OCP^O (38 μM) were monitored by TDA consisting of absorbance, refractive index, or right-angle light scattering detectors. The flow (0.5 mL/min) was split in two for TDA and SAXS detection, which is reflected in halved elution volumes shown on the X axis. The temperature was 20°C. Black and gray thick lines represent MW distributions over the Apo-OCP and OCP peaks, respectively. (B) SAXS curve (black) corresponding to the extreme left part of the Apo-OCP peak is presented on (A) with fits from crystallographic OCP^O monomer, OCP^O dimer, and a CORAL-derived model of the Apo-OCP dimer (see Materials and Methods for further details). (Inset) Given here is the resulting structural model of the Apo-OCP dimer superimposed with the corresponding ab initio envelope from the DAMMIF/DAMAVER procedure. (C) Corresponding structural models were drawn using PyMOL 1.6.9 (www.pymol.org). (D) Given here is approximation of the SAXS data for OCP^O obtained at high (black) or low (gray) protein concentration by the structural models presented on (C). (Inset) Models of the OCP^O monomer and dimer were superimposed with the corresponding ab initio envelopes from the DAMMIF/DAMAVER procedure. Color-coding is preserved throughout (B–D). Superposition of models with ab initio envelopes were made in the software UCSF Chimera v.1.11 (https://www.cgl.ucsf.edu/chimera/download.html) using the “fit to map” tool. To see this figure in color, go online.

Figure 4

Carotenoid transfer followed by SEC and native gel-electrophoresis. (A) Given here are SEC profiles of COCP and Apo-OCP, and of products of carotenoid transfer obtained by mixing COCP and Apo-OCP monitored by absorbance at indicated wavelengths. Note that dual wavelength detection allows the revealing of the spectral shift upon carotenoid transfer accompanying formation of OCP^O, whereas the fraction at ∼11.2 min has almost equal absorption at 460 and 560 nm. The OCP^O sample was loaded as the control. (Inset) Shown here is the color of COCP (the donor of carotenoid) and OCP^O (the product of transfer). (Dashed lines) Shown here are positions of the corresponding maxima of the peaks of Apo-OCP, COCP, and OCP^O. (B) Given here is SDS-PAGE analysis of the fraction obtained from the COCP profile (II) or its mixture with Apo-OCP after the completion of the carotenoid transfer (I). Fractions of the profile I are shown above the gel. SDS gels were stained by Coomassie brilliant blue. Positions of protein bands and those of MW markers are indicated. (C) The absorption spectra of the fraction was collected at ∼11.2 min in dark-adapted or light-adapted states and their difference spectrum is given, showing some photoactivity. (Dashed lines) Given here are characteristic spectral features. (D) Carotenoid transfer is followed by unstained native gel-electrophoresis. COCP was mixed with increasing amounts of Apo-OCP, incubated for 30 min at 33°C, and then loaded on the gel. Controls did not contain either COCP (first lane) or Apo-OCP (second lane). For details, see Materials and Methods. (Arrows) Given here are positions of carotenoid-containing proteins. To see this figure in color, go online.

Figure 5

Carotenoid transfer from COCP to GFP-OCP^Apo chimera. (A) Given here are absorption spectra of GFP-OCP chimera and related species. Upon addition of 4.4 μM of COCP (line 1) to a 6.3 μM solution of GFP-OCP^Apo (2), absorption of COCP gradually decreases. After equilibration of COCP-GFP-OCP^Apo interactions, the resulting spectrum of the system (3) represents the sum of GFP, OCP^O, and COCP absorption (dashed line). Obtained orange fraction is photoactive and, upon illumination of the sample by actinic light (450 nm, 200 MW), reversibly converts to the red state (4). Difference (5) between the spectra of the red and orange states is typical for all known OCP species. (B) Given here are the GFP fluorescence decay kinetics of GFP-OCP chimera in the absence (GFP-OCP^Apo) and in the presence of canthaxanthin (GFP-OCP^CAN). COCP to GFP-OCP^Apo ratio was equal to three. (Insets) Shown here is the structure of GFP (PDB:4EUL) and schematic representation of GFP-OCP chimera. (C) Given here are the kinetics of carotenoid transfer monitored by measurements of O.D. at 550 nm and intensity of GFP fluorescence at 510 nm, simultaneously. Experiment was conducted at 20°C and with constant stirring. To see this figure in color, go online.

Figure 6

Working model of the carotenoid transfer from COCP to Apo-OCP leading to reconstruction of a photoactive OCP. After mixing (Stage 0), COCP is anchored by Apo-OCP (presumably, via the NTE; Stage 1) and undergoes monomerization (critical; Stage 2) to transfer carotenoid into the NTD of Apo-OCP. Because the NTD has higher affinity to the carotenoid molecule than the CTD, it accepts the carotenoid from one of the CTD subunits of the anchored COCP. This leads to closure of OCP with bound carotenoid into the compact OCP^O-like structure (Stage 3) stabilized by carotenoid (Stage 4). Efficiency of carotenoid transfer is high, and >70% of carotenoid from COCP is transmitted to the orange photoactive form. However, as Apo-OCP tends to form homodimers at high concentrations, such structures could be stabilized by cross-domain carotenoid binding (confirmed by the data in Fig. 4). The alternative pathway (right part) requires preliminary monomerization of COCP (effectively achieved by COCP-W288A mutant) or involvement of some other carotenoid carrier. To see this figure in color, go online.