Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]

AMB Express

Table 1 Kinetic parameters for the binding of PhaR to DNAs and P(3HB) on the 27-MHz QCM ^a

Targets^b	DNA sequence^c	k _on ^d (10^-4 M^-1 s^-1)	k _off ^c (10^-3 s^-1)	K _d ^e (10^-7 M)
Control DNA^f	5′bio-TCGTTTAACGAGCCCGTATTTTCCCCTCTACCTTTTAGAGGACACCTAAC-3′	0.4	0.7	18
phaP1 promoter^g	5′bio-GGCGCATTTCTTATTTGGTGCGCCGCAACAATTCCTATTTTA GGGGCGCC-3′	6.0 ± 0.4	1.7 ± 0.4	3.2 ± 0.9
phaR promoter^f	5′bio-TCACGCGTTTAGCCATAGCGGGCGCGGTA GACGAACAACAGCACGGCCGG-3′	0.5	0.9	18
am-P(3HB)^g		7.0 ± 3.8	-^h	-^h

^a 10 mM HEPES buffer solution (pH 7.4) containing 150 mM NaCl and 0.002% Tween 20, 25°C. ^b 5′-biotinylated dsDNA immobilized on an avidin-covered QCM plate.
Amorphous-P(3HB) thin film was prepared by casting chloroform solution (1.0 wt%) of the isotactic P[(R), (S)-3-hydroxybutyrate]. ^c The bold type indicates the DNA binding site of PhaR revealed by DNase I footprinting (4). ^d k _on and k _off were obtained from Eq. 4. ^e K _d was calculated as k _off/k _on. ^f The data are from 2 independent experiments. ^g Amorphous P(3HB); the data are from 3 independent experiments. ^h K _d value could not be calculated due to a negative k _off value.