1. Volume 23, Issue 2, Pages 161-171 (July 2006)
Cyanobacterial Circadian Pacemaker: Kai Protein Complex Dynamics in the KaiC Phosphorylation Cycle In Vitro 
Hakuto Kageyama, Taeko Nishiwaki, Masato Nakajima, Hideo Iwasaki, Tokitaka Oyama, Takao Kondo 
Molecular Cell 
Volume 23, Issue 2, Pages (July 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
KaiC Phosphorylation Rhythm under Varying Concentrations of Kai Proteins
(A) KaiC phosphorylation rhythm in vitro under varying protein concentrations. Aliquots of the mixture taken at each time point were subjected to SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining or immunoblotting using an anti-KaiC antibody. P-KaiC and NP-KaiC designate phosphorylated and nonphosphorylated KaiC, respectively.
(B) Relative intensity of phosphorylated KaiC signals determined by densitometry of (A) are plotted against in vitro incubation time.
(C) KaiC phosphorylation rhythm under reduced KaiA concentrations (2/3 or 1/4 the concentration of the standard mixture). Data are labeled as in (B).
(D) KaiC phosphorylation rhythm under reduced KaiB concentrations (2/3 or 1/3 the concentration of the standard mixture). Data are labeled as in (B).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Association Profiles of Kai Proteins In Vitro
(A) Phosphorylation profile of KaiC, after incubation with KaiA-FLAG and KaiB-His6. At various times throughout the incubation, aliquot of the mixture (600 ng/lane) was subjected to SDS-PAGE, followed by CBB staining. P-KaiC and NP-KaiC designate phosphorylated and dephosphorylated KaiC bands, respectively.
(B) The phosphorylation level of KaiC was estimated by densitometric analysis of the data presented in (A) and plotted against the incubation time.
(C and D) Two-step pull-down assay. The mixtures of Kai proteins and ATP were collected at the indicated times throughout the in vitro KaiC phosphorylation cycle. Aliquots (50 μl) of each sample were initially subjected to pull-down assay with either anti-FLAG M2 beads (AC + ABC in [C]) or Ni-NTA agarose beads (BC + ABC in [D]). Then, the remaining supernatant was subjected to a second round of pull-down assays with the beads that had not been used in the first pull-down (BC in [C]; AC in [D]). The protein complexes isolated by each step were subjected to SDS-PAGE and stained with CBB. A 1/7.1 amount of the original solution was also analyzed to give a measure of the input KaiC (total KaiC) for estimation of proportion of precipitated KaiC (F). Total KaiC was prepared by mixing equal amounts of phosphorylated and nonphosphorylated KaiC, provided as described in Figure 4. Control indicates the negative control with recombinant wild-type KaiA and KaiB lacking C-terminal epitope tags. The results shown are representative of two independent experiments.
(E) The remaining supernatants of the second successive pull-down treatment were analyzed by SDS-PAGE (Figure S1); the free KaiC was plotted as the percentage of total KaiC. Open circles: first pull-down, KaiA-FLAG; second pull-down, KaiB-His6. Closed circle: first pull-down, KaiB-His6; second pull-down, KaiA-FLAG.
(F) The amounts of KaiC in the three types of complexes were estimated from densitometry of the data shown in (C) and (D) and expressed as a percentage of total KaiC. Open circles, closed circles, and open triangles indicate the amounts of KaiC in KaiA-KaiC (AC), KaiA-KaiB-KaiC (ABC), and KaiB-KaiC (BC) complexes, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Gel-Filtration Chromatography of the Standard Incubation Mixture throughout the KaiC Phosphorylation Cycle
Kai proteins were mixed under standard conditions and incubated at 30°C; then, 100 μl of aliquots were collected at the indicated incubation times (ITs). The protein samples were immediately applied at 4°C to Superose 6 columns with the AKTA Explorer. The protein contained in 18 fractions (11–20 ml elution volume) was precipitated with TCA (triaceticacid) and then subjected to SDS-PAGE with CBB staining. The graphs indicate densitometry of Kai proteins normalized to the elution volume. The vertical axes indicate the protein concentrations in each fraction.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Interaction Kinetics of the KaiA-KaiC and KaiB-KaiC Associations In Vitro
(A) Phosphorylated and dephosphorylated KaiC (see Experimental Procedures) was incubated at 30°C. Small fractions of KaiC were collected at the indicated times, followed by SDS-PAGE. P-KaiC and NP-KaiC designated phosphorylated and dephosphorylated KaiC bands, respectively.
(B and C) Phosphorylated (B) or dephosphorylated (C) KaiC (3.5 μM) was incubated at 30°C with KaiA-FLAG (1.2 μM). Aliquots of each protein mixture were collected at the indicated times and subjected to pull-down assay with anti-FLAG M2 beads. The isolated protein complexes were eluted with FLAG peptide (400 μg/ml), followed by SDS-PAGE. One-fifth of the amount of KaiC (phophorylated or dephosphorylated KaiC) in the original solution was used to estimate the KaiC input to allow determination of the relative proportions of KaiC in each complex. Control indicates the negative control performed with non-tagged KaiA. Input KaiC shows phosphorylation levels at each time point. Complex-associated KaiC amounts and KaiC phosphorylation levels were estimated by densitometry and plotted against incubation time over 24 hr. Open circles, proportion of complex-associated KaiC. Closed circles, phosphorylation state of input KaiC without pull-down. Results are presented as means ± standard deviation from three separate experiments.
(D and E) Phosphorylated (D) or dephosphorylated (E) KaiC (3.5 μM) was incubated at 30°C with KaiB-His6 (3.5 μM). Experimental procedures and annotations are the same as in (B) and (C), expect for using Ni-NTA and imidazole (500 mM) instead of anti-FLAG M2 beads and FLAG peptide, respectively. Results are presented as means ± standard deviation from three separate experiments.
(F and G) Pull-down assays were performed with phosphorylated KaiC, dephosphorylated KaiC, and KaiC[T432A;S431A] at indicated time points of the in vitro incubation. Isolated protein complexes were eluted, subjected to SDS-PAGE, and examined by CBB staining as described above.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Shuffling of KaiC Molecules between KaiC Hexamers
(A) A KaiC mixture (KaiC-FLAG: KaiC-His6=1:1, total KaiC was KaiC; 3.5 μM) was incubated for 0 or 3 hr, then subjected to Superose 6 column gel-filtration chromatography, as described in Figure 3. The 400–500 kDa signal corresponds to the KaiC hexamer.
(B) Schematic representation of experimental procedure testing the exchange of KaiC molecules among KaiC hexamers. Equimolar FLAG- and His6-tagged KaiC hexamers (KaiC mixture) were incubated and precipitated with anti-FLAG antibody. If KaiC molecules are completely exchanged between hexamers, the amount of KaiC precipitated with the FLAG antibody should increase 2-fold. If no exchange occurs, the precipitated KaiC would remain unchanged.
(C) KaiC mixture was incubated alone or in the presence of either KaiA (KaiA; 1.2 μM) or KaiB (KaiB; 3.5 μM) at 30°C. Aliquots were collected at the indicated times and precipitated with an anti-FLAG antibody. Immunoprecipitated proteins were subjected to 10% polyacrylamide gel electrophoresis to generate single-band signals, as described previously (Tomita et al., 2005). The densitometric intensities of precipitated KaiC were plotted against incubation time. The signals observed at the beginning of the incubation (0 hr) were normalized to 1.0. Results are presented as means ± standard deviation from three separate experiments.
(D) KaiC mixture (3.5 μM) was incubated in the presence of KaiA, and KaiB (KaiA, 1.2 μM; KaiB, 3.5 μM) was incubated at 30°C, then analyzed by anti-FLAG antibody immunoprecipitation as in (C). The densitometric intensity of the precipitated KaiC and the phosphorylation level of isolated KaiC were plotted against incubation time. Open circles indicate precipitated KaiC. The signals observed at the incubation start points (0 hr) were normalized to 1.0. Closed circles represent the phosphorylation level of KaiC. The shaded time period (4–8 hr) indicates the KaiC dephosphorylation phase.
(E) KaiC mixture using KaiC [S431A;T432A] was incubated alone or in the presence of either KaiA (KaiA, 1.2 μM) or KaiB (KaiB, 3.5 μM) at 30°C. Aliquots were collected, precipitated with an anti-FLAG antibody, and subjected to SDS-PAGE as described in (C).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 SPR Analysis of Kai Protein Associations
(A) Varying concentrations of KaiA (0.25, 0.5, 1, 2, and 4 μM) were injected onto immobilized KaiC. SPR units (RU) for each experiment were overlaid against the time after injection.
(B) Various concentrations of KaiB (2, 4, 6, 8, and 12 μM) in buffer A were injected onto immobilized KaiC. The responses are displayed as in (A). In both panels, buffer A with Kai protein was switched to plain buffer A (without Kai protein) at time 180 s (arrowhead) to monitor dissociations. The ka and kd values for these interactions were obtained by using the 1:1 Langmuir binding model with BIAevaluation 4.1 software of the Biacore system.
(C) KaiA (1 μM) was injected onto immobilized KaiC for various times (3, 30, and 60 min) and switched to the plain buffer A at time 0 (arrowhead). The dissociation of KaiA from immobilized KaiC was also monitored and shown as in (A). Experiments using varying KaiA concentrations (0.25, 0.5, and 2 μM) resulted in similar patterns.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 A Model for the KaiC Phosphorylation Cycle
The cycle of KaiC phosphorylation is illustrated as incubation time versus KaiC phosphorylation. The left and right frames indicate the principal protein dynamics for phosphorylation and dephosphorylation, respectively. See text for further explanation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions