Real-Time In Vitro Fluorescence Anisotropy of the Cyanobacterial Circadian Clock

Uniquely, the circadian clock of cyanobacteria can be reconstructed outside the complex milieu of live cells, greatly simplifying the investigation of a functioning biological chronometer. The core oscillator component is composed of only three proteins, KaiA, KaiB, and KaiC, and together with ATP they undergo waves of assembly and disassembly that drive phosphorylation rhythms in KaiC. Typically, the time points of these reactions are analyzed ex post facto by denaturing polyacrylamide gel electrophoresis, because this technique resolves the different states of phosphorylation of KaiC. Here, we describe a more sensitive method that allows real-time monitoring of the clock reaction. By labeling one of the clock proteins with a fluorophore, in this case KaiB, the in vitro clock reaction can be monitored by fluorescence anisotropy on the minutes time scale for weeks.


Introduction
Organisms from all domains of life display circadian (~24 h) rhythms in their metabolism, physiology, and behavior that arose as an adaptation to daily cycles of ambient light and temperature [1]. These endogenous rhythms are generated by intracellular circadian clocks. Despite extensive investigations in fungi, plants, insects, and vertebrates, the mechanistic nature of circadian clock protein-protein interactions remains mysterious. Among model systems, the cyanobacterial clock offers a unique opportunity in this regard. It can be reconstituted in vitro by simply mixing its three protein components-KaiA, KaiB, and KaiC-with ATP, resulting in a macroscopic~24 h rhythm of KaiC phosphorylation [2].
Just as a watchmaker's apprentice learns the mechanism of a watch by studying its gears as they move, it is informative to observe components of the cyanobacterial clock as they move. Typically, time points of in vitro cyanobacterial clock reactions are analyzed ex post facto using denaturing polyacrylamide gel electrophoresis (SDS PAGE) to resolve different states of KaiC phosphorylation. It was used to resolve the ordered temporal pattern of KaiC phosphorylation: S/T→S/pT→pS/pT→pS/T→S/T→ . . . , where S and T represent residues S431 and T432, the two phosphorylation sites of KaiC, and pS and pT denote their phosphorylated states [3,4]. This method has allowed for numerous insights into the cyanobacterial clock, such as KaiA stimulates KaiC autophosphorylation during the day, and KaiB inhibits KaiA in order to promote KaiC autophosphorylation during the day, and KaiB inhibits KaiA in order to promote KaiC autodephosphorylation at night [5][6][7][8][9][10]. However, it has some inherent disadvantages. For example, reactions cannot be monitored in real time. Removing aliquots from reactions every few hours followed by SDS PAGE limits how many experiments can be run in parallel and the duration of each experiment. Electrophoresis and densitometry of stained gels to determine KaiC phosphorylation levels is a manual process. In addition, this SDS PAGE approach has low temporal resolution (hour time scale), ≥10% uncertainty in KaiC phosphorylation levels per time point, does not directly inform on protein-protein interactions, and perturbs the sample (by taking time points). However, the recent development of an automated sampling device could reduce some of these drawbacks [11].
Here, we demonstrate that in vitro fluorescence spectroscopy can directly monitor circadian rhythms of protein-protein interactions in the cyanobacterial clock in real time by utilizing clock proteins labeled with fluorescent dyes. This methodology uses a fluorescently-labeled construct of KaiB [12][13][14], and the plasmid construct for expressing KaiB-FLAG-K25C (described in the Sections 2.1.1 and 3.1) is available to the scientific community. It is worth mentioning here that real-time measurement of luciferase-based bioluminescence in vivo is a powerful method for investigating circadian gene expression rhythms in cyanobacteria [15]. The availability of crystal structures of the free clock components and their complexes make it feasible to select fluorophore labeling sites that do not perturb the system [16]. As will be demonstrated below, tracking fluorescence anisotropy of 6-iodoacetamidofluroescein (6IAF) labeled KaiB allows direct observations of real-time population shifts between free KaiB (daytime) and bound KaiB (nighttime) (Figure 1). This fluorescence method is straightforward, does not perturb the sample during measurements, and offers a high time resolution (minutes) of the clock as it ticks. The methodology utilizes standard molecular cloning, protein expression, and labeling procedures. A convenient approach to site-directed DNA mutagenesis is Quikchange PCR, allowing for substitutions, deletions, or small additions with a single-step polymerase chain reaction conducted on a plasmid vector [18]. Then, standard heat shock transformation of E. coli, expression, purification of the protein and fluorophore labeling sets the stage for facile real-time measurements of protein-protein interactions in oscillating clock reactions. Figure 2 provides structural insight into the selected KaiB conjugation site for fluorophore labeling, highlighting how lysine 25 is oriented away from the intramolecular tetramer and dimer interfaces, and intermolecular KaiC and KaiA binding sites, allowing for conserved function of KaiB.
Fluorescence anisotropy is widely used by biochemists. Basically, shining polarized light on an isotropic solution selectively excites fluorophores whose transition moments are parallel to the The methodology utilizes standard molecular cloning, protein expression, and labeling procedures. A convenient approach to site-directed DNA mutagenesis is Quikchange PCR, allowing for substitutions, deletions, or small additions with a single-step polymerase chain reaction conducted on a plasmid vector [18]. Then, standard heat shock transformation of E. coli, expression, purification of the protein and fluorophore labeling sets the stage for facile real-time measurements of protein-protein interactions in oscillating clock reactions. Figure 2 provides structural insight into the selected KaiB conjugation site for fluorophore labeling, highlighting how lysine 25 is oriented away from the intramolecular tetramer and dimer interfaces, and intermolecular KaiC and KaiA binding sites, allowing for conserved function of KaiB.   Fluorescence anisotropy is widely used by biochemists. Basically, shining polarized light on an isotropic solution selectively excites fluorophores whose transition moments are parallel to the direction of polarization [19]. Excited-state lifetimes of many commercially available fluorophores are similar to the rotational correlation times of proteins (nanoseconds), making fluorescence anisotropy of labeled proteins sensitive to protein-protein interactions ( Figure 1).  Figure 3, with 300 W xenon arc lamp attenuated such that fluorescently-labeled samples yield 600,000-800,000 photon counts, was used for all fluorescence measurements described here. This steady-state fluorimeter was setup with a three-stage cuvette system and water bath to control sample temperature. Excitation and emission wavelengths are easily manipulated, allowing for measurements of multiple fluorescent dyes simultaneously.

Experimental
• Stock MgCl2 and ATP solutions. Critical step: ATP and MgCl2 stocks at concentrations of 300 mM and 200 mM, respectively, were used. These should be prepared in 20 mM Tris with pH 8.0. Note that KaiA and KaiB protein stocks lack ATP and MgCl2. • 1.5 mL sterile centrifuge tubes, three for each reaction sample (see steps 80-88).  [21]. Here, once the sample is excited by polarized light, only the right instrument path is utilized, with the right photon multiplier tube collecting both parallel and perpendicular emissions in turn through software-controlled emission polarizer orientation adjustments (bottom right purple rectangle).  [21].
Here, once the sample is excited by polarized light, only the right instrument path is utilized, with the right photon multiplier tube collecting both parallel and perpendicular emissions in turn through software-controlled emission polarizer orientation adjustments (bottom right purple rectangle).

•
Circulating water bath at 30 • C and tubing compatible with fluorimeter for sample temperature control.
centrifuge at 13,000 rpm for 1 min. 4. Incubate Quikchange PCR products with DpnI for 1 hour at 37 °C. 5. PCR purification kit, optional (QUIAGEN). 6. Measure Quikchange PCR product concentration with Nanodrop. Ensure the 260 nm/280 nm ratio is near 1.8 for pure DNA product. Critical step: Sample purity can be obtained by comparing the absorbance at 260 nm against that of 280 nm (A260/280). Highly pure DNA in should be ~1.8, while highly pure RNA should be ~2.0. Proteins absorb light at 280 nm. Therefore, lower ratios are indicative of protein contamination in the sample [22].    Critical step: The PCR products should be run on 1.5% agarose gels before performing DpnI digestion. Load 3 µL PCR product and 3 µL 6× orange loading dye. Mix well with micropipette and heat to 100 • C for 1 min before loading agarose gel. Only add DpnI to the products that show bright bands at the correct size of the plasmid.

3.
Add 1 µL of DpnI enzyme to each Quikchange PCR product, and spin down in benchtop centrifuge at 13,000 rpm for 1 min.
Measure Quikchange PCR product concentration with Nanodrop. Ensure the 260 nm/280 nm ratio is near 1.8 for pure DNA product. Critical step: Sample purity can be obtained by comparing the absorbance at 260 nm against that of 280 nm (A 260/280 ). Highly pure DNA in should be~1.8, while highly pure RNA should be~2.0. Proteins absorb light at 280 nm. Therefore, lower ratios are indicative of protein contamination in the sample [22].
Add 50 µL of BL21-DE3 competent cells to each chilled centrifuge tube. 10. Add 2 µL of DpnI digested Quikchange PCR product, mix well and incubate on ice for 30 min.
Critical step: For the final five minutes of the 30 min period in which the Quikchange PCR product is on ice, place the 2× YT media broth stock tubes in the hot water bath at 42 • C. Remove the 2× YT media broth from the hot water bath at the same time the heat shock ends (see step 11). 11. Heat shock the cells at 42 • C in a water bath for 45 s. 12. Incubate them on ice for 2 min. 13. Add 500 µL of warmed 2× YT broth to each sample.
14. Incubate cells in temperature-controlled shaker at 37 • C and 220 rpm for 1 h. 15. Spin down cells at 5000 rpm for 1 min in benchtop centrifuge (at room temperature). 16 1 µL incubated transformation product will be used in step 24, and the remained of the cultured 5 mL LB-kan + broth media may be stored at 4 • C. This culture will be utilized following gel electrophoresis analysis to ensure proper DNA length (see step 29). 24. Prepare PCR centrifuge tubes with 1 µL incubated transformation product, 1 µL forward primer (10 µM), 1 µL reverse primer (10 µM), 10 µL dNTP (10 mM), 1 µL Pfu Turbo DNA Polymerase (1 unit/50 µL), 5 µL 10× Pfu Turbo Cx Reaction Buffer, and dilute to 50 µL with deionized water (see Table 1). 25. Run PCR thermo cycle to amplify transformed DNA sequence (see Figure 4). 26. Prepare a 1.5% agarose gel. 27. Load 30 µL of PCR product into 1.5% agarose gel, along with 1 kbp DNA ladder, and run at 60 V for 20 min. 28. Analyze PCR product bands to confirm that the DNA sequence is both present at a reasonable concentration and the correct length. 29. Spin down remainder of cultures for 10 min at 4 • C and 4000 rpm using Thermo Scientific Sorvall Legend RT+ Centrifuge. 30. Discard supernatant and isolate DNA plasmid from pelleted cells using Thermo Scientific GeneJET Plasmid Miniprep Kit. 31. Measure DNA miniprep product with Nanodrop. Expect concentration of 100-300 ng/µL. 32. Prepare 500 ng of miniprep product in deionized water (10 µL total volume each). 33. Each sample requires two tubes, one with forward primer (0.5 µL at 10 µM) and the other with reverse primer (0.5 µL at 10 µM) added. 34. Send samples to DNA sequencing facility. 35. Analyze results to ensure desired mutations are present before moving forward with protein expression.

LB Media (Time of Completion: 2 h)
36. Add 25 g LB to 1 L of deionized water in 2 L Pyrex flask. Critical step: LB media should be used for KaiA and KaiB-FLAG expression. For FLAG-KaiC, M9 minimal media is highly recommended.
To produce 1 L of M9 media, mix 6 g Na 2 HPO 4 , 3 g KH 2 PO 4 , 0.5 g NaCl, and 1 g NH 4 Cl, then autoclave on liquid cycle. Following sterilization, add 10 mL (0.2 µm filtered) 20% D-glucose, 2 mL 1 M MgSO 4 , 100 µL 1 M CaCl 2 , and 1 mL 50 µg/mL kanamycin. 37. Autoclave on a liquid cycle to sterilize media (121 • C for 20 min). 38. Allow to cool to 50 • C, and add 1 mL (50mg/mL) kanamycin to the sterilized 1 L LB media. 51. Transfer the cell lysate into 50 mL centrifuge tubes and spin down at 15,000 rpm for 45 min. 52. Prepare Ni-NTA columns for purification by running two full column volumes of deionized water through the nickel-chelated resin, followed by equilibration with a full column volume of lysis buffer. 53. Load supernatant onto Ni-NTA columns. Critical step: Preceding the SUMO tag is a 6-His-tag that binds the Ni 2+ ions, trapping the proteins of interest on the Ni-NTA column. 54. Wash column with 50 mL wash buffer (50 mM NaH 2 PO 4 , 500 mM NaCl, 20 mM imidazole, pH 8.0) to remove non-His tagged proteins. 55. Elute His tagged protein with 5 mL elution buffer (50 mM NaH 2 PO 4 , 500 mM NaCl, 250 mM imidazole, pH 8.0). Critical step: It is advised that 20 µL aliquots of supernatant, wash, and eluate be collected and analyzed by SDS-PAGE following Ni-NTA purification. This can help to quickly determine whether the protein of interest is in the eluate (desirable) or wash (undesirable). 56. Add 150 µL of 100 µM ubiquitin-like-specific-protease 1 (Ulp1). Critical step: The Ulp1 enzyme specifically cleaves His 6 -SUMO tag from the fusion protein, allowing for purification by subsequent passes through Ni-NTA columns in which the His 6 -SUMO tag binds while the protein does not. 57. Allow 8-14 h for Ulp1 to cleave His 6 -SUMO fusion proteins at 4 • C. 58. Dilute the solution to 25 mM imidazole by adding lysis buffer to the sample. 59. Clean Ni-NTA columns with two column volumes of DI water, followed by two bed volumes of 0.2 µm-filtered solution of 6 M guanidine hydrochloride (GdHCl) in 0.2 M acetic acid. Then wash with another two column volumes of DI water. 60. Pass Ulp1-treated protein samples through clean Ni-NTA columns, and collect the flow-through in a clean, labeled corning tube. Critical step: When passing Ulp1-treated protein samples through Ni-NTA columns, it is important to regulate flow rates to ensure that the free His 6 -SUMO fusion tag can be captured on the Ni-NTA column, while proteins of interest pass through. This was performed by attaching a 1-2" section of soft rubber tubing to the bottom of each Ni-NTA column, which can then be partially clamped to allow flow rates of~50-60 drop/min. 61. Repeat steps 59 & 60 to ensure that protein sample has been sufficiently separated from His 6 -SUMO tags. Critical step: After cleavage of His 6 -SUMO by Ulp1 and purification by Ni-NTA columns, a confirmation step can be performed to verify that the protein of interest is present and pure. Transfer 3-5 µL of nickel column eluate to a centrifuge tube and add an equal volume of 2× SDS-dye. Load the sample alongside a protein ladder on SDS polyacrylamide gel (17% for KaiB) and run for 30 min at 60 V, followed by 100 min at 120 V. A comparison of stained bands against molecular weight markers allow for the estimation of protein purity and yield. 62. Concentrate purified samples to 6 mL for subsequent FPLC injection (see Section 3.4.5) in an Amicon Stirred Cell concentrator using a 10 kDa molecular weight cut-off (MWCO) membrane.

Fast Protein Liquid Chromatography (Time of Completion: 2-3 h)
63. Prepare mobile phase buffer in which the purified protein will be stored (20 mM Tris, 150 mM NaCl, pH 8, or pH 7.0 if the protein will be fluorescently labeled). Critical step: The preferred ratio of 6IAF to protein is 10-20:1, although we have had success at 5:1 fluorescently labeling KaiB. During the conjugation reaction the recommended pH is 7.0-7.5, which is accomplished by preparing the mobile phase for FPLC at this pH. All labeling was performed at 4 • C overnight in the dark. 64. Divide the 6 mL of concentrated protein sample into 1 mL aliquots in six 1.5 mL centrifuge tubes and spin down at 13,000 rpm for 2 min on a benchtop centrifuge. This will allow for removal of any precipitate and particles from the sample before FPLC. 65. Draw the supernatant from the centrifuge tubes into a 10 mL syringe using a long blunt-end needle.
66. Exchange the needle on the syringe with an adaptor for connecting to the FPLC sample injection port. 67. Remove all bubbles from the syringe. 68. Attach the syringe to the sample injection port on the FPLC. Do so without introducing air into the instrument by filling the injection port with DI water as the syringe adaptor is inserted. 69. Run the FPLC with HiLoad 16/600 Superdex 75 size-exclusion column, at 0.8 mL/min flow rate, and 0.5 MPa maximum pressure. 70. Collect eluate peak fractions for proteins of interest. 71. Following purification, concentrate eluate to desired concentration with an Amicon Stirred Cell concentrator using a 10 kDa molecular weight cut-off (MWCO) membrane. During the 30-min timer duration, prepare the fluorimeter for data collection. Here, using 6IAF as a reporter, the monochromators on the PC1 excitation and emission wavelengths of 492 nm (slit width 2.0 nm) and 520 nm (slit width 1.0 nm), respectively, were read every 15 min. Fluorescence anisotropy measurements are achieved on the PC1 with an emission polarizer isolating parallel emission detection followed by subsequent perpendicular emission detection with the same photomultiplier tube (PMT). G-factor determination is set to the "once" option to adjust parallel and perpendicular gain values in order to eliminate instrumental bias in fluorescence anisotropy measurements. 87. Load clock reaction samples into 1 mL syringe, attach 0.2 µm membrane filter, and filter samples into fresh 1.5 mL centrifuge tubes. Critical step: Filtering the solutions with low protein-binding 0.2 µm membrane ensures bacteria and/or large contaminants are not present in the final reaction mixtures. This step may be omitted if long term oscillations, many days/weeks, are not planned. 88. Transfer into a fresh 1.5 mL centrifuge tube the volume of sample necessary to achieve a final volume of 600 µL upon adding fluorescently labeled protein in step 89. 89. In the dark, add KaiB-FLAG-K25C-fluorophore to a final concentration of 0.05 µM and total volume of 600 µL. 90. In the dark, mix the sample and transfer it to a fluorimeter cuvette. 91. Place the cuvette in the fluorimeter and start data collection once the 30-min timer (see step 86) expires. Record the time representing t = 0. Figure 5 shows real-time fluorescence anisotropy measurements of KaiB-FLAG-K25C-fluorophore over the course of 12 days. Note that robustness of the oscillation indicates that the experiment could have been extended considerably longer. Trough anisotropies are when labeled KaiB is mostly free in solution. In contrast, peak anisotropies are when KaiB is bound to KaiC and KaiA. The stability of the troughs is likely due to the stability of labeled KaiB. The slow decrease of peak anisotropies is likely due to a steady decrease in ATP and functional KaiC protein levels. In order to minimize potential artifacts due to the fluorophore, only 1.4% (50 nM) KaiB was labeled. Fluorescence intensities over 12 days decreased from 720,000 counts to 670,000, indicating that photobleaching is not a significant concern. A significant advantage of this protocol is that it allows hundreds of in vitro reactions to be monitored simultaneously in real time using a plate reader with minutes time resolution for weeks (this protocol has been employed to monitor oscillations on the Tecan Spark 10M and BMG CLARIOstar plate readers with nearly identical results relative to data collected on the PC1). Assuming that 15 ATP molecules are consumed per day per KaiC particle [23], after 12 days the ATP concentration is expected to be 0.3-0.4 mM. Note that the oscillator begins to fail around 0.1 mM ATP [23]. Although only KaiB was labeled in this protocol, KaiA and KaiC can in principle also be fluorescently labeled, although care must be taken regarding their naturally occurring cysteinyl residues.

Expected Results
Critical step: Filtering the solutions with low protein-binding 0.2 µ m membrane ensures bacteria and/or large contaminants are not present in the final reaction mixtures. This step may be omitted if long term oscillations, many days/weeks, are not planned. 88. Transfer into a fresh 1.5 mL centrifuge tube the volume of sample necessary to achieve a final volume of 600 µ L upon adding fluorescently labeled protein in step 89. 89. In the dark, add KaiB-FLAG-K25C-fluorophore to a final concentration of 0.05 µ M and total volume of 600 µ L. 90. In the dark, mix the sample and transfer it to a fluorimeter cuvette. 91. Place the cuvette in the fluorimeter and start data collection once the 30-min timer (see step 86) expires. Record the time representing t=0. Figure 5 shows real-time fluorescence anisotropy measurements of KaiB-FLAG-K25Cfluorophore over the course of 12 days. Note that robustness of the oscillation indicates that the experiment could have been extended considerably longer. Trough anisotropies are when labeled KaiB is mostly free in solution. In contrast, peak anisotropies are when KaiB is bound to KaiC and KaiA. The stability of the troughs is likely due to the stability of labeled KaiB. The slow decrease of peak anisotropies is likely due to a steady decrease in ATP and functional KaiC protein levels. In order to minimize potential artifacts due to the fluorophore, only 1.4% (50 nM) KaiB was labeled. Fluorescence intensities over 12 days decreased from 720,000 counts to 670,000, indicating that photobleaching is not a significant concern. A significant advantage of this protocol is that it allows hundreds of in vitro reactions to be monitored simultaneously in real time using a plate reader with minutes time resolution for weeks (this protocol has been employed to monitor oscillations on the Tecan Spark 10M and BMG CLARIOstar plate readers with nearly identical results relative to data collected on the PC1). Assuming that 15 ATP molecules are consumed per day per KaiC particle [23], after 12 days the ATP concentration is expected to be 0.3-0.4 mM. Note that the oscillator begins to fail around 0.1 mM ATP [23]. Although only KaiB was labeled in this protocol, KaiA and KaiC can in principle also be fluorescently labeled, although care must be taken regarding their naturally occurring cysteinyl residues.