Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]
© Yamada et al.; licensee Springer. 2013
Received: 25 December 2012
Accepted: 22 January 2013
Published: 27 January 2013
The repressor protein PhaR, which is a component of poly[(R)-3-hydroxybutyrate] granules, functions as a repressor of the gene expression of the phasin PhaP and of PhaR itself. We used a quartz crystal microbalance to investigate the binding behavior by which PhaR in Ralstonia eutropha H16 targets DNAs and amorphous poly[(R)-3-hydroxybutyrate] thin films. Binding rate constants, dissociation rate constants, and dissociation constants of the binding of PhaR to DNA and to amorphous poly[(R)-3-hydroxybutyrate] suggested that PhaR bind to both in a similar manner. On the basis of the binding rate constant values, we proposed that the phaP gene would be derepressed in harmony with the ratio of the concentration of the target DNA to the concentration of amorphous poly[(R)-3-hydroxybutyrate] at the start of poly[(R)-3-hydroxybutyrate] synthesis in R. eutropha H16.
KeywordsPolyhydroxyalkanoate Autoregulator protein PhaR Kinetic analysis Ralstonia eutropha H16
Polyhydroxyalkanoate (PHA), an eco-friendly and biodegradable polyester, is synthesized by a variety of bacteria, as their intracellular storage material for carbon and energy (Doi et al. 1995; Steinbuchel and Fuchtenbusch 1998; Sudesh et al. 2000). In bacterial cells, PHA forms granules that are covered with a layer composed of proteins and phospholipids (Potter et al. 2002). The most abundant constituent of this layer is phasin (PhaP). The presence of PhaP on the surface of PHA granules contributes to the reduction in size of PHA granules as well as to the slight enhancement of PHA production (Kojima et al. 2006; Potter et al. 2002; Potter and Steinbuchel 2005). Recently, the ability of PhaP to bind to a hydrophobic surface was used to develop methods for protein purification, drug delivery, and tissue engineering applications in in vitro experiments (Backstrom et al. 2007; Banki et al. 2005; Wang et al. 2008). In the cells of microorganisms, a repressor protein PhaR regulates the expression of phaP and phaR. PhaR has also been reported to sense the presence of PHA and to interact with nascent PHA granules, resulting in the derepression of phaP expression (Potter et al. 2002; Potter and Steinbuchel 2005). The presence of genes homologous to PhaR and PhaP in the genomes of various PHA-producing bacteria suggests that a similar regulatory system by PhaR is likely to exist in PHA-producing bacteria (Eugenio et al. 2010; Kojima et al. 2006; Maehara et al. 2002; Yamada et al. 2007; Yamashita et al. 2006). This regulatory system of PHA production through phaR and phaP expression can be applied in a two-hybrid system for protein-protein interaction (Wang et al. 2011). Therefore, understanding of the regulatory system provides meaningful benefit to not only basic science but also applications in various fields such as industry and medicine.
In previous studies, the binding behaviors of PhaR to target DNA (including the promoter region of phaP) and to melt-crystallized thin films of poly[(R)-3-hydroxybutyrate] [cr-P(3HB)] were investigated using surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) measurements (Yamada et al. 2007; Yamashita et al. 2006). However, kinetic parameters such as the binding rate constant (k on) and dissociation rate constant (k off) by which PhaR targets DNA and P(3HB) have not been determined thus far. These kinetics and stoichiometric analyses will contribute new insights into the behavior of PhaR in the regulatory system of phaP expression. In order to determine the precise kinetic parameters, we selected a multichannel QCM sensing system to monitor the binding reaction of PhaR from Ralstonia eutropha H16 to target DNAs (including the promoter regions of phaP and phaR) and thin films of amorphous P(3HB) [am-P(3HB)] derived from atactic P(3HB). This is because the P(3HB) native granule is composed of am-P(3HB). Recently, the regulatory system of PHA production through phaR and phaP expression has been applied in studies of protein-protein interaction, protein purification, drug delivery, and tissue engineering. The insights gained into this regulation mechanism in this study have the potential to improve applications in white biotechnology. We have determined kinetic parameters based on mass changes on the DNA-immobilized and am-P(3HB)-coated QCM oscillators, and discuss the binding behavior of PhaR with target DNA and am-P(3HB).
Expression and purification of autoregulator protein PhaR
Calibration of 27-MHz QCM in aqueous solution
The QCM apparatus was an AFFINIX Q4 (Initium Co., Ltd., Tokyo, Japan) with 4 500-μL cells equipped with a 27-MHz QCM plate (8.7 mm diameter quartz plate and 5.7 mm2 area Au electrode) at the bottom of the cell and a stirring bar with a temperature control system (Takahashi et al. 2007; Takahashi et al. 2008). The relationship between mass and frequency changes in aqueous solutions when DNAs and/or proteins were immobilized onto the QCM was calibrated by comparing it against values in the air phase. One Hz of frequency represents a 0.10 ng cm-2 mass increase on the QCM plate. The noise level of the 27-MHz QCM was ±2 Hz in buffer solutions at 25°C, and the stability of the frequency was ±2 Hz for 1 h in buffer at 25°C.
Preparation of the DNA-Immobilized QCM Oscillator
Kinetic parameters for the binding of PhaR to DNAs and P(3HB) on the 27-MHz QCM a
k on d (10-4 M-1 s-1)
k off c (10-3 s-1)
K d e (10-7 M)
6.0 ± 0.4
1.7 ± 0.4
3.2 ± 0.9
7.0 ± 3.8
Preparation of P(3HB) thin films
QCM oscillators were washed with a freshly prepared Piranha solution of H2O2/H2SO4 (1/3 v/v) and were rinsed several times with Milli-Q water. (Caution: Piranha solution is very oxidative and dangerous, and direct contact should be avoided). Thin films of P(3HB) were prepared on the QCM oscillators by casting 300 μl of chloroform solutions (1.0–1.5 wt%) of the polymers on a spin-coater at 4000 rpm under dry air.
Reactions in the DNA-immobilized or am-P(3HB) coated QCM oscillator
Enzyme reactions in a DNA-immobilized or am-P(3HB) coated QCM cell were performed with 500 μL of assay buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.002% Tween 20). The frequency changes in response to the addition of enzymes were then followed over time. The solution was vigorously stirred to avoid any effects from the slow diffusion of the enzymes. The stirring did not affect the stability or magnitude of the frequency changes.
In order to understand the regulatory system governing PHA production in detail, we investigated the binding behaviors of PhaR to the target DNA (containing the promoter region of phaP) and to P(3HB), using QCM measurements. Regarding PhaR-DNA binding, Figure 2A shows that PhaR mainly bound to the DNA containing the phaP promoter region (curve a), and barely bound to the DNA containing the phaR promoter region (curve b) or to the control DNA (curve c). The binding curve of PhaR to the phaP promoter region showed sigmoid curve, implying that PhaR binds to target DNA in a cooperative reaction. The SPR analysis of PhaR-DNA binding in previous studies was not capable of monitoring the initial binding of PhaR, because the concentration of PhaR (10 μM) was higher than in the present experimental conditions (2.5 to 10 nM) (Kojima et al. 2006; Maehara et al. 2002). The higher binding affinity of PhaR to the phaP promoter region accorded with the results of gel-mobility-shift assays (Maehara et al. 2002). The DNA fragments with the phaP promoter region shifted at a lower concentration of PhaR compared to the DNA fragments that contained the phaR promoter region (Potter et al. 2002).
The binding rate constant for the DNA containing the phaR promoter region (k on = 0.5 × 104 M-1 s-1) was similar to the parameters for the control DNA (k on = 0.4 × 104 M-1 s-1) (Table 1). Moreover, the dissociation rate constant for the DNA containing the phaP promoter region (k off = 1.7 ± 0.4 × 10-3 s-1) was not significantly different from the dissociation rate constant for the DNA containing the phaR promoter region (k off = 0.9 × 10-3 s-1) or that of the control DNA (k off = 0.7 × 10-3 s-1). These parameters indicate that PhaR had higher affinity for the phaP promoter region than for the phaR promoter region. The larger k on value for the DNA with the phaP promoter region must have been due to the length of the recognition sequence for PhaR in the target DNA region. A 32-bp region TGC-rich sequence is recognized by PhaR in the phaP promoter region, while the phaR promoter region included only an 8-bp recognition sequence (Table 1) (Potter and Steinbuchel 2005). On the basis of the k on values obtained in this study, the difference in k on values between the promoter regions of phaP and phaR corresponds to the hypothetical model of PhaR-mediated phaP expression (Maehara et al. 2002; Potter and Steinbuchel 2005; Yamada et al. 2007). In particular, when PHA is not accumulated in the cells, the presence of PhaR is necessary to repress the gene expression of phaP. Since PhaP is a predominantly PHA granule-associated protein, PhaP production is not required for the cells without PHA accumulation (Maehara et al. 2002). Thus, the lower k on value for DNA with the phaR promoter region indicates weak repression of phaR expression in cells.
In the measurement of PhaR-am-P(3HB) binding, we did not obtain k off and K d values of the binding of PhaR to am-P(3HB), because the k off was negative. This result indicates that the binding of PhaR to am-P(3HB) is an irreversible interaction (Table 1). There was no significant difference between the k on value for am-P(3HB) (k on = 7.0 ± 3.8 × 104 M-1 s-1) and that for the DNA containing the phaP promoter region (k on = 6.0 ± 0.4 × 104 M-1 s-1), which implied that the derepression of phaP expression was prompted by an increase in the concentration of am-P(3HB) in the cells. In other words, the concentration-dependent effect was one of the main factors initiating the expression of phaP at the onset of the dissociation of PhaR from the phaP promoter region in cells.
In conclusion, we observed initial binding behaviors between PhaR and target molecules such as target DNAs and am-P(3HB), using QCM techniques. Based on the QCM data, kinetic parameters (k on, k off, and K d) for the binding of PhaR to target molecules were determined by the kinetic analysis of obtained binding curves. These values provided a novel insight into the binding behavior of PhaR with target molecules. The phaP gene is likely derepressed in harmony with the ratio of the concentration of the target DNA to the concentration of am-P(3HB) at the beginning of P(3HB) synthesis in microbes. On the basis of the results of a previous paper (Maehara et al. 2002), we assumed that PhaR dissociates from the PhaR/DNA complex when P(3HB) is accumulated under intracellular conditions. This finding indicates that the effector molecules of PhaR are P(3HB) molecules. Also, one of the factors responsible for the dissociation of PhaR from DNA is the high affinity of PhaR to P(3HB). The binding of PhaR to DNA and to am-P(3HB) showed similar k on values, suggesting that a concentration-dependent effect caused the expression of phaP with dissociation of PhaR from the phaP promoter region. The insights of the regulation mechanism concerning PhaR in PHA synthesis have the potential to improve the applications of PHA in white and red biotechnology.
Quartz crystal microbalance
- (k on):
Binding rate constant
- (k off):
Dissociation rate constant
- (K d):
Sodium dodecyl sulfate
Polyacrylamide gel electrophoresis.
The authors declare no competing financial interest. Supported by RIKEN Biomass Engineering Program.
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