Photosynthetic reaction centers are sensitive to high light conditions, which can cause damage because of the formation of reactive oxygen species. the same crystal. Based on amino acid conservation analysis, activity assays of FRP mutants, FRP:OCP docking simulations, and coimmunoprecipitation experiments, we conclude that this dimer is the active form. The second form, a tetramer, may be an inactive form of FRP. In addition, we have identified a surface patch of highly conserved residues and shown that those residues are essential to FRP activity. conformation (3C6). Irradiance with high light changes the OCP from an inactive orange (OCPo) to an active red form (OCPr) that is capable of binding to the phycobilisomes to prevent excess energy from flowing to the reaction centers. The low quantum yield of the OCPo to OCPr photoconversion together with instability of the OCPr form lead to effective OCP inactivity under low light Tropisetron HCL supplier conditions; therefore, the OCP acts as a switch for photoprotection triggered by a specific light level (7). In darkness, isolated OCPr spontaneously reverts back to the OCPo form. This reversion is usually greatly affected by the presence of the fluorescence recovery protein (FRP) (8). In vitro, the FRP accelerates the conversion of free OCPr back to the orange form. In vivo, the FRP is essential to recover the full capacity of the antenna, presumably by playing a role in detaching OCPr from the phycobilisomes (8). Mutant strains of cyanobacteria lacking this protein PTGS2 are unable to recover the normal antenna capacity under low light conditions. The FRP is a 13 kDa protein that does not bind a chromophore. It is exclusively found in organisms that also contain the OCP, and the two genes are typically in close proximity in the genome. Of the currently available 130 cyanobacterial genomes, 97 genomes contain a gene for the OCP, and 71 genomes also contain a gene for the FRP (9) (Table S1). The FRP from sp. PCC 6803 was first characterized in a form containing an additional 25 aa compared with the FRP from almost all other strains Tropisetron HCL supplier (the other exception is the FRP encoded in the genome) (8). It has recently been shown that this longer form is caused by a misidentified start site, and the active form in vivo is a shorter protein that begins with Met26 (10); we have used Tropisetron HCL supplier this form for this study. Here, we present the 3D structure of the FRP; the protein crystallized in two different conformations and Tropisetron HCL supplier different quaternary says. We show that this active form of the FRP is a homodimer, with a cluster of highly conserved residues on one surface of the dimer. Based on these observations, we made several single amino acid mutations, analyzed the mutant forms for activity, and thereby, identified the active site of the FRP. Based on the structural information, docking, and coimmunopreciptation studies, we propose a model for the conversation between the FRP and the OCP. These results provide the foundation for additional studies around the molecular mechanism of the regulation of photoprotection in cyanobacteria. Results Structure of the FRP Shows That It Adopts Two Distinct Conformations. We have decided the crystal structure of sp. PCC 6803 FRP at 2.5 ? resolution using iodine phasing (Table S2). There are six FRP polypeptide chains in the asymmetric unit of the P41212 space group in two distinct conformations. Four (chains ACD) form dimers, with the twofold axis either in the asymmetric unit or generated by crystal symmetry. Remarkably, the other two chains (chains E and F) in the asymmetric unit exist in a very different conformation: -helices 1 and 2 form one long extended helix 1, making a dimer that, together with its symmetry-related chains (E and Tropisetron HCL supplier F), forms a four-helix bundle (Fig..