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DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy ab

c

bcd

Sung Hyun Kim , So Young Kim , Kwang-Hwan Jung

abd

& Doseok Kim

a

Department of Physics, Sogang University, Seoul, Korea

b

Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, Korea

c

Department of Life Science, Sogang University, Seoul, Korea

d

Institute of Biological Interfaces, Sogang University, Seoul, Korea Published online: 10 Mar 2015.

Click for updates To cite this article: Sung Hyun Kim, So Young Kim, Kwang-Hwan Jung & Doseok Kim (2015) DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy, Bioscience, Biotechnology, and Biochemistry, 79:7, 1070-1074, DOI: 10.1080/09168451.2015.1015950 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1015950

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Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 7, 1070–1074

DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy Sung Hyun Kim1,2, So Young Kim3, Kwang-Hwan Jung2,3,4 and Doseok Kim1,2,4,* 1

Department of Physics, Sogang University, Seoul, Korea; 2Interdisciplinary Program of Integrated Biotechnology, Sogang University, Seoul, Korea; 3Department of Life Science, Sogang University, Seoul, Korea; 4Institute of Biological Interfaces, Sogang University, Seoul, Korea

Received August 5, 2014; accepted January 20, 2015

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http://dx.doi.org/10.1080/09168451.2015.1015950

Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, KD, of ASRT to 20 bp-long DNA fragments lied in micromolar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination. Key words:

fluorescence; fluorescence correlation spectroscopy; DNA–protein interaction; Anabaena sensory rhodopsin transducer; photo-signal transduction

Anabaena sensory rhodopsin (ASR) is a membrane protein that senses light in cyanobacteria and triggers cellular responses depending on the light environment.1) It is believed that the ASR signal transduction cascade regulates the expression of various proteins related to the light-harvesting system, the circadian clock, and the chromatic adaptation.2) The mechanism of photo-transduction of ASR is, however, not fully understood yet. ASR expresses together with a 14 kDa transducer (Anabaena sensory rhodopsin transducer, ASRT) in a single operon.3) ASRT is a soluble tetrameric form4) and has been found to interact with ASR in the cell membrane.2,3) Thus, it is suggested that ASRT transduces the signal from ASR to the cytoplasm of the cell.1) While the details of the signal transduction cascade remains to be explored, regulation of the several related

genes is suggested as the functional role of ASRT due to its binding ability to DNA.5,6) Proteins structurally homologous to ASRT are found in clusters with the encoding proteins involved in sugar metabolism, as well as with other membrane proteins related to bioenergetics.4,7) Also, ASRT is a member of a novel superfamily of β-sandwich fold domain including the sugar-binding site.3,4,7) These β-sandwich structures are also found in several transcription factors of immunoglobulin-like fold. This kind of DNA-binding proteins takes roles as important developmental, immunological, and anticancer regulators.3) Thus, ASRT may be a representative of a new class of transducers that interact with DNA on one end and with membrane proteins and/or small metabolites on the other end. In this study, we applied fluorescence correlation spectroscopy (FCS) to characterize the interaction of ASRT with DNA. Since its development in early 1970s, FCS has been adapted in various fields such as physics, chemistry, and biology.8–13) FCS measures diffusive motion of fluorescent molecules in solution to obtain their thermodynamic properties such as diffusion coefficient and hydrodynamic radius.14–18) In biology, FCS has been used to investigate interactions between biomolecules such as DNA and proteins labeled with fluorescent dye molecules.19–24) Upon binding of other molecules, the effective size of the fluorescently labeled molecule increases and, as a result, the diffusion coefficient decreases. In this report, we utilized the capability of FCS in detecting and identifying the change of diffusive motion of different fluorescent species in solution to study nucleoprotein complex formation between ASRT and DNA. In our previous study, by means of various biochemical methods, we showed evidences of DNA binding activity as a potential gene regulator.6) Here, we elucidate and characterize the molecular details of ASRT/DNA interaction with different sequences of possible targets in the signal cascade or with random/artificial sequences.

*Corresponding author. Email: [email protected] Abbreviations: ASR, Anabaena sensory rhodopsin; ASRT, Anabaena sensory rhodopsin transducer; BSA, bovine serum albumin; FCS, fluorescence correlation spectroscopy; PEC, phycoerythrocyanin. © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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Materials and methods Details of the FCS setup are described in our previous report.25,26) Briefly, a confocal microscopy setup was built around an inverted microscope (Olympus, IX51) equipped with an objective lens (Olympus, NA 1.2, water immersion), a 532 nm laser (Crystalaser) and an avalanche photodiode (PerkinElmer). We used a commercial counting system (National Instrument, NI6602) for real-time recording of photon detection events from which the time-correlation function was calculated on the fly using home-built software written in LabView (National Instrument). Typically, fluorescence photons were collected for 180 s at a count rate higher than 104 Hz. The confocal volume of the setup was calibrated using a reference fluorescence dye (TAMRA, Invitrogen). All FCS measurements were carried out in a buffer consisting of 10 mM TrisHCl (pH 8.0), 50 mM NaCl, 100 pM of DNA oligomer and ASRT of which concentration is specified in the text and figure caption. All measurements were performed at room temperature. ASRT was prepared as described in our previous report.27) Overnight cultures of Escherichia coli BL21 that contains the plasmid for the over-production of his-tagged ASRT proteins were induced with 0.8 mM IPTG (Duchefa) for 4 h at 35 °C. The induced E. coli cells were harvested and suspended in sonication buffer (50 mM TrisHCl pH 7.0, 150 mM NaCl). The cells were lysed with 0.5 mM PMSF (phenylmethylsulfonyl fluoride, USB) by sonication (Branson sonifier 250) at 4 °C followed by low-speed (3220 g for 20 min) centrifugation (Eppendorf centrifuge 5810R) to remove cell debris. Cell lysates were then sedimented at 95,000 × g for 1 h at 4 °C (Ti70 rotor, Beckman XL-90 ultracentrifuge), and the supernatant was transferred to a new tube. The supernatant was incubated with Ni2+–NTA agarose (Qiagen) and eluted with imidazole. Typically, we obtained 30 mg of ASRT from the culture of 500 mL of induced E. coli cells. All the fluorescently labeled DNA oligomers were purchased from IDTDNA and used without further purification. The sequences designated as R2 and R3 (see Table 1) were selected as follows: we aligned with other ASRT binding promoters and selected four most conserved regions and tested for gel shift assay (Supplementary Fig. 1). Among the four regions, R2, R3, and R4 regions showed better binding than R1. For FCS measurements, we have chosen R2 and R3 as these two regions showed the highest and moderate binding affinities to ASRT, respectively. The Amp

Table 1. Name *

R2 R3** Rand20 Amp*** PolyA

DNA sequences. Sequence 5′-GCAATCGACCTTATAAAAAG-3′ 5′-ATGACCTTTAGGAGGAAAGA-3′ 5′-TACCTCGGAGGCAATAGCTT-3′ 5′-GCGGTTAGCTCCTTCGGTCC-3′ 5′-AAAAAAAAAAAAAAAAAAAA-3′

*R2 sequence was selected from the pec promoter region (−55 to −36). **R3 sequence was selected from the pec promoter region (−30 to −11). ***Amp sequence was selected from the ampicillin gene (+427 to +446).

Fig. 1. Normalized correlation functions of fluorescence-labeled DNA (R3). Notes: (a) Correlation functions measured at different concentrations of ASRT (symbols) with the best fits to the data (solid lines). Inset: magnification of the middle region of the correlation functions. (b) Correlation functions measured with (red) and without (black) 100 μM BSA. Inset: magnification of the middle region of the correlation functions.

sequence was selected from the ampicillin gene (+427 to +446). The Rand20 sequence was a random sequence carrying equal numbers of the four bases.

Results and discussion To characterize interaction between ASRT and DNA oligomers, we measured correlation functions of the fluorescently labeled DNA oligomers. We first selected a sequence of a DNA fragment (R3, Table 1) from a small region of the promoter of the gene phycoerythrocyanin (pec), because the gene is involved in the chromatic adaptation of cyanobacteria, which is a potential target of ASRT in the signal transduction pathway.6) The 20 bp sequence was chosen from the last 100 bp of the pec promoter region, as the region was reported to have higher DNA binding activity.6) To obtain the hydrodynamic radius of bare DNA in our reaction buffer condition (10 mM TrisHCl pH 8.0, 50 mM NaCl), we measured the DNA oligomers in the absence of ASRT. The measured correlation function (Fig. 1(a), black rectangles) showed a single decay in the sub-millisecond regime and was fitted well by the analytic solution of the correlation function of a single-species free diffusion model28):

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GðsÞ ¼

1 \N [



1 1  4Ds =r02



S. H. Kim et al.

1 1  4Ds =z20

1=2

:

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(1) Here, is the average number of the fluorescence dye molecules in the confocal detection volume of which the lateral and longitudinal lengths are r0 and z0, respectively. The determined diffusion coefficient, D, of the DNA oligomer (R3) was 62 μm2/s. According to the Stokes–Einstein relation (D = kBT/6πηr, where kB is Boltzmann’s constant, T is the absolute temperature, η is the viscosity, and r is the hydrodynamic size of the fluorescent species), the hydrodynamic radius of the oligomer was estimated to be 3.5 nm in our experimental condition. This value seems reasonable as it corresponds to one-half of the full extension of a 20 bp-long B-form DNA. Note that the hydrodynamic radius is estimated under the assumption of globular shape of the object, as we are interested in detecting the relative shifts of the diffusion coefficients to study the protein/ DNA interaction, but are less concerned about the exact shape of the object. We then carried out FCS measurements at different concentrations of ASRT (0–200 μM), while keeping the concentration of the DNA at 1 nM. Shown in Fig. 1(a) are selected correlation functions measured at different concentrations of ASRT. The correlation functions were normalized by its amplitude at zero time delay G(0) to visualize the shift of the correlation functions at different ASRT concentrations. Inset in Fig. 1(a) magnifies the middle region of the correlation functions, which clearly shows the shift of the correlation functions to a longer time regime with the increase of ASRT concentration. The shift indicates building-up of large-sized fluorescent species in the solution. In our experimental condition, because the fluorescence dye molecule was attached to DNA, the size increase can be achieved only by the formation of DNA–ASRT complex. Note that a correlation function obtained in the presence of a similar-size (or larger) protein (bovine serum albumin, BSA) did not show any difference to that of bare DNA (Fig. 1(b)), ruling out the possibility that the shift observed in Fig. 1(a) is from an experimental artifact such as crowding effect or viscosity change by the protein. For the quantitative analysis, we fit the correlation functions with two-species model (one for bare DNA and the other for DNA–ASRT complex) and extracted out the diffusion coefficient and the fraction of DNA oligomers that are bound to ASRT10):   1=2 m 1X 1 1 Fi : GðsÞ ¼  N i 1 þ 4Di s=r02 1 þ 4Di s=z20

diffusion coefficient of the DNA–ASRT complex was determined as 19 μm2/s, which corresponds to the hydrodynamic radius of the complex of 11.4 nm. All the correlation functions at different ASRT concentrations were fitted globally by fixing the above two diffusion coefficient to yield Fi’s in Equation (2) at different concentrations. The determined size of the complex is ~3 times the size of the tetrameric structure solved by crystallography (3.5 nm in radius).4) The 20 bp DNA oligomer has a rod-like shape with 6.8 nm in length and 2.0 nm in diameter, and a fluorescence dye is attached at the end of the DNA via flexible liker. The size of DNA–ASRT complex estimated from above is a bit smaller than the observed hydrodynamic radius, presumably an irregular shape of the DNA–ASRT complex worked to increase the hydrodynamic radius. We further characterized the binding affinity of ASRT with five different DNA sequences (Table 1). The sequences were adapted from another region of the pec operon promoter (R2) or from a small region in the middle of the ampicillin gene (Amp). We also examined artificial sequences such as a random DNA sequence (Rand20) or a poly adenine sequence (PolyA). Correlation functions obtained from the rest of the sequences (Supplementary Fig. 2) also showed similar shift in the presence of ASRT at different concentrations as that observed with R3. Shown in Fig. 2 are the fractions of DNA–ASRT complex at different concentrations of ASRT. As expected, the fraction of the DNA–ASRT complex increased with the ASRT concentration. By fitting the fractions with the Hill equation we obtained the dissociation constants for the five different sequences as shown in Fig. 3: F ¼ Fmax 

½ASRT : KD þ ½ASRT

(3)

Here, F is the bound fraction of the oligomers and KD is the dissociation constant. We assumed that only one DNA molecule could bind to ASRT because the

(2) Here, Fi is the number fraction of the species i, is the average number of the total fluorescent molecules in the focal volume, and the summation (to m) is over the fluorescent species present in the solution. The fitted functions are depicted as solid lines in Fig. 1. During the fitting, we fixed the diffusion coefficient of bare DNA to the value obtained above (62 μm2/s). The

Fig. 2. The fraction bound obtained from the five different sequences listed in Table 1. Notes: Filled box: Amp (sequence adapted from ampicillin gene), open circle: R3 (from pec promoter), open triangle: R2 (from pec promoter), filled diamond: PolyA (A tract), and cross: Rand20 (random sequence). Solid lines are the best fits to the Hill equation. Error bars are standard deviations from three independent sets of ASRT concentration-dependent FCS measurements.

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sensitivity of FCS measurement, which allowed us to examine the biomolecules in action. We showed preferential binding affinity of ASRT to certain DNA sequences. This observation supports the idea that ASRT has the regulatory role in gene expression related to the light-harvesting system.

Author contribution

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Fig. 3. The dissociation constant, KD, of ASRT on different DNA sequences. Note: Error bars are standard errors from three independent sets of ASRT concentration-dependent FCS measurements.

concentration of the DNA was significantly lower (more than three orders) than that of ASRT in our measurement such that the probability to have two DNA on a single ASRT was very low. The measured dissociation constants were in the range of 6–17 μM and varied with the substrate sequences (Fig. 3). For example, the largest KD measured with the Rand20 sequence was ~2.7 times larger than that obtained with the Amp sequence. An unpaired-samples t-test concluded that there was a significant difference between the measured KD of amp and Rand20; t = 7.081, p = 0.002. t-Test results on the other sequences were summarized on Supplementary Table 1. This sequence-dependent binding affinity change of ASRT to DNA suggests that specific genes are the targets of ASRT in the downstream signal transduction pathways. Thus, the genes related to the chromatic adaptation system can be directly regulated by physical interaction of ASRT to the promoter regions of the genes. Interestingly, the Amp sequence showed highest binding activity, whereas a 280 bp sequence containing the Amp sequence (20 bp) was found not to interact with ASRT in our previous report.6) This observation suggests that an ASRT tetramer may bind to a longer region of DNA than 20 bp, or to several non-consecutive regions from a DNA. This is conceivable because it is suggested that ASRT works as repressor and activator like AraC in a way that it is not a single binding site that is influencing the expression but the other binding sites are also important for inducing bending of this promoter region.6) A related note is that there are only moderate changes in the measured binding affinity partially because the DNA fragments we examined are relatively short: 20 bp-long DNA fragments. Due to the practical limitation of FCS technique, we were not able to examine longer DNA substrates. But, in vivo, ASRT may interact with longer region of the DNA, to result in more specific sequence dependence. This moderate change in affinity also suggests that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of genes. In summary, we applied FCS technique to investigate the binding activity of ASRT, a transducer protein involved in the photo-signal transduction cascade in cyanobacteria. We took advantage of the high

SHK, SYK, KJ and DK conceived the study. SHK, SYK performed the measurements. SHK, SYK analyzed the data. SHK, SYK, KJ, DK wrote the manuscript.

Supplementary material The supplementary material for this paper is available at http://10.1080/09168451.2015.1015950.

Acknowledgments This work was supported by the Sogang University Research Grant of 2014 (201410043.01). KHJ acknowledges support by National Nuclear R&D Program funded by the Ministry of Science ICT & future planning (2012055325).

Conflict of interest The authors declare that they have no conflict of interest.

Funding This work was supported by the Sogang University Research Grant of 2014 [grant number 201410043.01]. KHJ acknowledges support by National Nuclear R&D Program funded by the Ministry of Science ICT & future planning [grant number 2012055325].

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DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy.

Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena...
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