Soluble domains of cytochrome c-556 and Rieske iron-sulfur protein from Chlorobaculum tepidum: Crystal structures and interaction analysis.

In photosynthetic green sulfur bacteria, the electron transfer reaction from menaquinol:cytochrome c oxidoreductase to the P840 reaction center (RC) complex occurs directly without any involvement of soluble electron carrier protein(s). X-ray crystallography has determined the three-dimensional structures of the soluble domains of the CT0073 gene product and Rieske iron-sulfur protein (ISP). The former is a mono-heme cytochrome c with an α-absorption peak at 556 nm. The overall fold of the soluble domain of cytochrome c-556 (designated as cyt c-556sol) consists of four α-helices and is very similar to that of water-soluble cyt c-554 that independently functions as an electron donor to the P840 RC complex. However, the latter's remarkably long and flexible loop between the α3 and α4 helices seems to make it impossible to be a substitute for the former. The structure of the soluble domain of the Rieske ISP (Rieskesol protein) shows a typical ß-sheets-dominated fold with a small cluster-binding and a large subdomain. The architecture of the Rieskesol protein is bilobal and belongs to those of b6f-type Rieske ISPs. Nuclear magnetic resonance (NMR) measurements revealed weak non-polar but specific interaction sites on Rieskesol protein when mixed with cyt c-556sol. Therefore, menaquinol:cytochrome c oxidoreductase in green sulfur bacteria features a Rieske/cytb complex tightly associated with membrane-anchored cyt c-556.

Energy transfer fluctuation observed by single-molecule spectroscopy of red-shifted bacteriochlorophyll in the homodimeric photosynthetic reaction center.

The photosynthetic reaction center of heliobacteria (hRC) is a homodimeric chromoprotein responsible for light harvesting and photoelectric conversion. The fluorescence of the hRC is radiated from a bacteriochlorophyll (Bchl) g having the lowest energy level, called red-Bchl g. The homodimeric architecture of the hRC indicates that it includes two red-Bchls g arranged symmetrically in pairs. Red-Bchl g is a fluorescent probe useful for monitoring the energy transfer network in the RC. Here, we show the fluorescence polarization dependences of two red-Bchls g, individually measured with selective excitation of chlorophyll a serving as the primary electron acceptor. The two red-Bchls g exhibit almost the same polarization dependences. Based on the polarization dependence and structural data of the hRC, we propose a candidate molecule for red-Bchl g. The fluorescence spectra of single hRCs represent the spectral heterogeneity reflecting the local conformational inhomogeneity. A time series of the fluorescence spectra indicates occasional peak shifts between blue- and red-shifted states without significant changes in the fluorescence intensity. The spectral fluctuation is interpreted to be due to the local conformational dynamics around a Bchl g mediating the energy transfer, switching the terminal energy acceptor between two red-Bchls g. In conclusion, while the energy transfer network in the RC can be perturbed by microscopic dynamics, the total energy transfer efficiency, i.e., the light-harvesting function, is rather robust. The functional robustness may be due to multiple energy transfer pathways composed of many antenna pigments in the RC.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy.

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

Crystal structure of higher plant heme oxygenase-1 and its mechanism of interaction with ferredoxin.

Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.

Rhythmic adenosine triphosphate release from the cyanobacterial circadian clock protein KaiC revealed by real-time monitoring of bioluminescence using firefly luciferase.

The cyanobacterial circadian clock is composed of three clock proteins, KaiA, KaiB and KaiC. This KaiABC clock system can be reconstituted in vitro in the presence of adenosine triphosphate (ATP) and Mg2+ , and shows circadian rhythms in the phosphorylation level and ATPase activity of KaiC. Previously, we found that ATP regulates a complex formation between KaiB and KaiC, and KaiC releases ATP from KaiC itself (PLoS One, 8, 2013, e80200). In this study, we examined whether the ATP release from KaiC shows any rhythms in vitro. We monitored the release of ATP from wild-type and ATPase motif mutants of KaiC as a bioluminescence in real time using a firefly luciferase assay in vitro and obtained the following results: (a) ATP release from KaiC oscillated even without KaiA and KaiB although period of the oscillation was not 24 hr; (b) ATP was mainly released from the N-terminal domain of KaiC; and (c) the ATP release was enhanced and suppressed by KaiB and KaiA, respectively. These results suggest that KaiC can generate basal oscillation as a core clock without KaiA and KaiB, whereas these two proteins contribute to adjusting and stabilizing the oscillation.

The role of ROC75 as a daytime component of the circadian oscillator in Chlamydomonas reinhardtii.

The circadian clocks in chlorophyte algae have been studied in two model organisms, Chlamydomonas reinhardtii and Ostreococcus tauri. These studies revealed that the chlorophyte clocks include some genes that are homologous to those of the angiosperm circadian clock. However, the genetic network architectures of the chlorophyte clocks are largely unknown, especially in C. reinhardtii. In this study, using C. reinhardtii as a model, we characterized RHYTHM OF CHLOROPLAST (ROC) 75, a clock gene encoding a putative GARP DNA-binding transcription factor similar to the clock proteins LUX ARRHYTHMO (LUX, also called PHYTOCLOCK 1 [PCL1]) and BROTHER OF LUX ARRHYTHMO (BOA, also called NOX) of the angiosperm Arabidopsis thaliana. We observed that ROC75 is a day/subjective day-phase-expressed nuclear-localized protein that associates with some night-phased clock genes and represses their expression. This repression may be essential for the gating of reaccumulation of the other clock-related GARP protein, ROC15, after its light-dependent degradation. The restoration of ROC75 function in an arrhythmic roc75 mutant under constant darkness leads to the resumption of circadian oscillation from the subjective dawn, suggesting that the ROC75 restoration acts as a morning cue for the C. reinhardtii clock. Our study reveals a part of the genetic network of C. reinhardtii clock that could be considerably different from that of A. thaliana.

Cryogenic Single-Molecule Spectroscopy of the Primary Electron Acceptor in the Photosynthetic Reaction Center.

The photosynthetic reaction center (RC) converts light energy into electrochemical energy. The RC of heliobacteria (hRC) is a primitive homodimeric RC containing 58 bacteriochlorophylls and 2 chlorophyll as. The chlorophyll serves as the primary electron acceptor (Chl a-A0) responsible for light harvesting and charge separation. The single-molecule spectroscopy of Chl a-A0 can be used to investigate heterogeneities of the RC photochemical function, though the low fluorescence quantum yield (0.1%) makes it difficult. Here, we show the fluorescence excitation spectroscopy of individual Chl a-A0s in single hRCs at 6 K. The fluorescence quantum yield and absorption cross section of Chl a-A0 increase 2- and 4-fold, respectively, compared to those at room temperature. The two Chl a-A0s in single hRCs are identified as two distinct peaks in the fluorescence excitation spectrum, exhibiting different excitation polarization dependences. The spectral changes caused by photobleaching indicate the energy transfer across subunits in the hRC.

Electron transport pathways in isolated chromoplasts from Narcissus pseudonarcissus L.

During daffodil flower development, chloroplasts differentiate into photosynthetically inactive chromoplasts having lost functional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase (PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b6 f complex, ATP synthase and several isoforms of ferredoxin-NADP+ oxidoreductase (FNR), and ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b6 f complex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors for the NDH complex, the cytochrome b6 f complex, FNR and redox-inactive Fd in which the iron was replaced by gallium. Our data suggest an electron flow via two separate pathways, both reducing plastoquinone (PQ) and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd and cytochrome bh of the cytochrome b6 f complex, and does not result in the pumping of protons across the membrane. In the second, electron transport takes place via the NDH complex using both NADH and NADPH as electron donor. FNR and Fd are not involved in this pathway. The NDH complex is responsible for the generation of the proton gradient. We propose a model for chromorespiration that may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b6 f complex during cyclic electron transport in chloroplasts.

X-ray structure of an asymmetrical trimeric ferredoxin-photosystem I complex.

Photosystem I (PSI), a large protein complex located in the thylakoid membrane, mediates the final step in light-driven electron transfer to the stromal electron carrier protein ferredoxin (Fd). Here, we report the first structural description of the PSI-Fd complex from Thermosynechococcus elongatus. The trimeric PSI complex binds three Fds in a non-equivalent manner. While each is recognized by a PSI protomer in a similar orientation, the distances between Fds and the PSI redox centres differ. Fd binding thus entails loss of the exact three-fold symmetry of the PSI's soluble subunits, inducing structural perturbations which are transferred to the lumen through PsaF. Affinity chromatography and nuclear magnetic resonance analyses of PSI-Fd complexes support the existence of two different Fd-binding states, with one Fd being more tightly bound than the others. We propose a dynamic structural basis for productive complex formation, which supports fast electron transfer between PSI and Fd.

X-ray crystallographic and high-speed AFM studies of peroxiredoxin 1 from Chlamydomonas reinhardtii.

Peroxiredoxins (PRXs) are a group of antioxidant enzymes that are found in all organisms, including plants and green algae. The 2-Cys PRX from Chlamydomonas reinhardtii (CrPRX1) is a chloroplast-localized protein that is critical for clearing reactive oxygen species in chloroplasts. CrPRX1 is reduced by thioredoxins or calredoxin (CrCRX), a recently identified calcium-dependent redox protein. The molecular interaction between PRXs and thioredoxin/CrCRX is functionally important, but discussion has been limited owing to a lack of structural information on CrPRX1, especially regarding its oligomeric state. In this study, high-speed atomic force microscopy (HS-AFM) images of CrPRX1 and an X-ray crystallographic analysis have enabled examination of the oligomeric state of CrPRX1. Diffraction data from a crystal of the Cys174Ser mutant of CrPRX1 indicate the existence of noncrystallographic fivefold symmetry. HS-AFM images of CrPRX1 further show that CrPRX1 particles form rings with pentagonal rotational symmetry. On the basis of these findings, the oligomeric state of CrPRX1 is discussed and it is concluded that this PRX exists in a ring-shaped decameric form comprising a pentamer of dimers.

Association of Ferredoxin:NADP+ oxidoreductase with the photosynthetic apparatus modulates electron transfer in Chlamydomonas reinhardtii.

Ferredoxins (FDX) and the FDX:NADP+ oxidoreductase (FNR) represent a key junction of electron transport downstream of photosystem I (PSI). Dynamic recruitment of FNR to the thylakoid membrane has been considered as a potential mechanism to define the fate of photosynthetically derived electrons. In this study, we investigated the functional importance of the association of FNR with the photosynthetic apparatus in Chlamydomonas reinhardtii. In vitro assays based on NADP+ photoreduction measurements as well as NMR chemical shift perturbation analyses showed that FNR preferentially interacts with FDX1 compared to FDX2. Notably, binding of FNR to a PSI supercomplex further enhanced this preference for FDX1 over FDX2, suggesting that FNR is potentially capable of channelling electrons towards distinct routes. NADP+ photoreduction assays and immunoblotting revealed that the association of FNR with the thylakoid membrane including the PSI supercomplex is impaired in the absence of Proton Gradient Regulation 5 (PGR5) and/or Proton Gradient Regulation 5-Like photosynthetic phenotype 1 (PGRL1), implying that both proteins, directly or indirectly, contribute to the recruitment of FNR to the thylakoid membrane. As assessed via in vivo absorption spectroscopy and immunoblotting, PSI was the primary target of photodamage in response to high-light stress in the absence of PGR5 and/or PGRL1. Anoxia preserved the activity of PSI, pointing to enhanced electron donation to O2 as the source of the observed PSI inactivation and degradation. These findings establish another perspective on PGR5/PGRL1 knockout-related phenotypes and potentially interconnect FNR with the regulation of photosynthetic electron transport and PSI photoprotection in C. reinhardtii.

Dynamics and energetics of cyanobacterial photosystem I:ferredoxin complexes in different redox states.

Fast turnover of ferredoxin/Fd reduction by photosystem-I/PSI requires that it dissociates rapidly after it has been reduced by PSI:Fd intracomplex electron transfer. The rate constants of Fd dissociation from PSI have been determined by flash-absorption spectroscopy with different combinations of cyanobacterial PSIs and Fds, and different redox states of Fd and of the terminal PSI acceptor (FAFB). Newly obtained values were derived firstly from the fact that the dissociation constant between PSI and redox-inactive gallium-substituted Fd increases upon (FAFB) reduction and secondly from the characterization and elucidation of a kinetic phase following intracomplex Fd reduction to binding of oxidized Fd to PSI, a process which is rate-limited by the foregoing dissociation of reduced Fd from PSI. By reference to the complex with oxidized partners, dissociation rate constants were found to increase moderately with (FAFB) single reduction and by about one order of magnitude after electron transfer from (FAFB)- to Fd, therefore favoring turnover of Fd reduction by PSI. With Thermosynechococcus elongatus partners, values of 270, 730 and >10000s-1 were thus determined for (FAFB)Fdoxidized, (FAFB)-Fdoxidized and (FAFB)Fdreduced, respectively. Moreover, assuming a conservative upper limit for the association rate constant between reduced Fd and PSI, a significant negative shift of the Fd midpoint potential upon binding to PSI has been calculated (< -60mV for Thermosynechococcus elongatus). From the present state of knowledge, the question is still open whether this redox shift is compatible with a large (>10) equilibrium constant for intracomplex reduction of Fd from (FAFB)-.

Hyperfine Sublevel Correlation Spectroscopy Studies of Iron-Sulfur Cluster in Rieske Protein from Green Sulfur Bacterium Chlorobaculum tepidum.

The magnetic properties of the Rieske protein purified from Chlorobaculum tepidum were investigated using electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy (HYSCORE). The g-values of the Fe2S2 center were gx = 1.81, gy = 1.90, and gz = 2.03. Four classes of nitrogen signals were obtained by HYSCORE. Nitrogens 1 and 2 had relatively strong magnetic hyperfine couplings and were assigned as the nitrogen directly ligated to Fe. Nitrogens 3 and 4 had relatively weak magnetic hyperfine couplings and were assigned as the other nitrogen of the His ligands and peptide nitrogen connected to the sulfur atom via hydrogen bonding, respectively. The anisotropy of nitrogen 3 reflects the different spin density distributions on the His ligands, which influences the electron transfer to quinone.

Gallium ferredoxin as a tool to study the effects of ferredoxin binding to photosystem I without ferredoxin reduction.

Reduction of ferredoxin by photosystem I (PSI) involves the [4Fe-4S] clusters FA and FB harbored by PsaC, with FB being the direct electron transfer partner of ferredoxin (Fd). Binding of the redox-inactive gallium ferredoxin to PSI was investigated by flash-absorption spectroscopy, studying both the P700+ decay and the reduction of the native iron Fd in the presence of FdGa. FdGa binding resulted in a faster recombination between P700+ and (FA, FB)-, a slower electron escape from (FA, FB)- to exogenous acceptors, and a decreased amount of intracomplex FdFe reduction, in accordance with competitive binding between FdFe and FdGa. [FdGa] titrations of these effects revealed that the dissociation constant for the PSI:FdGa complex is different whether (FA, FB) is oxidized or singly reduced. This difference in binding, together with the increase in the recombination rate, could both be attributed to a c. -30 mV shift of the midpoint potential of (FA, FB), considered as a single electron acceptor, due to FdGa binding. This effect of FdGa binding, which can be extrapolated to FdFe because of the highly similar structure and the identical charge of the two Fds, should help irreversibility of electron transfer within the PSI:Fd complex. The effect of Fd binding on the individual midpoint potentials of FA and FB is also discussed with respect to the possible consequences on intra-PSI electron transfer and on the escape process.

Circadian oscillations of KaiA-KaiC and KaiB-KaiC complex formations in an in vitro reconstituted KaiABC clock oscillator.

The circadian clock is an endogenous biological mechanism that generates autonomous daily cycles in physiological activities. The phosphorylation levels of KaiC oscillated with a period of 24 h in an ATP-dependent clock oscillator reconstituted in vitro from KaiA, KaiB and KaiC. We examined the complex formations of KaiA and KaiB with KaiC in the KaiABC clock oscillator by fluorescence correlation spectrometry (FCS) analysis. The formation of KaiB-containing protein complex(es) oscillated in a circadian manner, with a single peak at 12 h and single trough at 24 h in the circadian cycle, whereas that of KaiA-containing protein complex(es) oscillated with two peaks at 12 and 24 h. FCS and surface plasmon resonance analyses showed that the binding affinity of KaiA for a mutant KaiC with Ala substitutions at the two phosphorylation sites considered to mimic the nonphosphorylated form of KaiC (np-KaiC) was higher than that for a mutant KaiC with Asp substitutions at the two phosphorylation sites considered to mimic the completely phosphorylated form of KaiC (cp-KaiC). The results from the study suggest that a KaiA-KaiB-cp-KaiC ternary complex and a KaiA-np-KaiC complex were formed at 12 and 24 h, respectively.

Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast.

Calcium (Ca(2+)) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca(2+)- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca(2+)-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca(2+)-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca(2+)-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca(2+)-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.

Bacterial ß-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2).

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 ß-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.

X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin.

In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the [2Fe-2S] cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Šresolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.

Expression and crystallization of a bacterial glycoside hydrolase family 116 ß-glucosidase from Thermoanaerobacterium xylanolyticum.

The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS-PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pNP) glycosides pNP-ß-D-glucoside, pNP-ß-D-galactoside and pNP-N-acetyl-ß-D-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 M sodium citrate tribasic, 0.1 M Tris-HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions of 120×25×5 µm. The TxGH116 crystals diffracted X-rays to 3.15 Šresolution and belonged to the monoclinic space group P2(1). Structure solution will allow a structural explanation of the effects of human GBA2 mutations.

Importance of the monomer-dimer-tetramer interconversion of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.

The molecular machinery of the cyanobacterial circadian clock oscillator consists of three proteins, KaiA, KaiB and KaiC, which interact with each other to generate circadian oscillations in the presence of ATP (the in vitro KaiABC clock oscillator). KaiB comprises four subunits organized as a dimer of dimers. Our previous study suggested that, on interaction with KaiC, the tetrameric KaiB molecule dissociates into two molecules of dimeric KaiB. It is uncertain whether KaiB also exists as a monomer and whether the KaiB monomer can drive normal circadian oscillation. To address these questions, we constructed a new KaiB oligomer mutant with an N-terminal deletion, KaiB10-108 . KaiB10-108 was a monomer at 4 °C but a dimer at 35 °C. KaiB10-108 was able to drive normal clock oscillation in an in vitro reconstituted KaiABC clock oscillator at 25 °C, but it was not able to drive normal circadian gene expression rhythms in cyanobacterial cells at 41 °C. Wild-type KaiB existed in equilibrium between a dimer and tetramer at lower KaiB concentrations or in the presence of 1 m NaCl. Our findings suggest that KaiB is in equilibrium between a monomer, dimer and tetramer in cyanobacterial cells.