2A10
Exploring excited state properties of BLUF domain of AppA by photothermal methods
P. Hazra1, K. Inoue1, W. Laan2, K. J. Hellingwerf2, and M. Terazima1
Kyoto University1, Swammerdam Institute for Life Sciences, The Netherlands2
Introduction:
AppA is a novel blue-light receptor that regulates photosynthesis gene expression in response to blue
light in the purple bacterium Rhodobacter sphaeroides [1,2]. AppA interacts with a transcriptional repressor
PpsR to form a stable AppA-(PpsR)2 complex in the dark or lowlight conditions [1,2]. Blue light disrupts thebinding of AppA to PpsR and thus restoring the PpsR binding activity to the promoter region of photosynthesis
genes [1,2]. Although AppA mediated signal transduction is rather well understood, its initial photochemistry
and signal transduction pathway still remain to be solved. Since the time-resolved transient grating (TG)technique has been well established to detect kinetics and conformational change or aggregation process which
may occur far away from chromophore, we investigated AppA (BLUF domain of AppA, AppA5-125)
photoreaction after the blue light irradiation by the time-resolved TG method. We have also used the transientlens (TrL) method for quantitative measurements of the enthalpy change of the final product.

Experimental:
The principle of TG and TrL techniques are described elsewhere [3-5]. Our excitation wavelength inboth TG and TrL experiments was 465 nm (XeCl excimer laser pumped dye laser). The probe wavelength for
the TG measurement was 780 nm (from diode laser) and the probe wavelength for the TrL measurement was633 nm (from He-Ne laser). BLUF domain of AppA, AppA5-125 was cloned, and expressed from Escherichia
coli [6].

Results and Discussions:
The TG signal of AppA upon the photoexcitation rose quickly with the time response of our system(~20 ns) and there appeared a weak slow rising component with a time constant of ~3.4 µs, which was
attributed to the decay of the excited triplet state. After this component, the thermal grating signal and finally arise-decay component representing protein diffusion process were observed. From the rate constants of the riseand decay of the latest signal, the diffusion coefficient of AppA and the product can be determined.
Interestingly, we found that this component was concentration dependent in all q2 condition (Fig.1). In goingfrom low q2 to high q2 condition, the signal approaches to a single exponential like feature. We interpreted thisconcentration dependent feature in terms of the aggregation formation in the excited state of this protein. Theobserved TG signal was fitted well by an aggregation model. The observed aggregation rate constant (kobs)
increases linearly as the concentration increases. This dependence suggests that the dimerization reaction takes
place in the photoreaction of AppA. From the slope of the plot of kobs against the concentration, we got second
order rate constant with a value ~2.5×105 M-1sec-1. This value is lower than the diffusion controlled rate

observed for bimolecular association reaction.
Furthermore, we have measured the enthalpy change associated with the formation of the firstintermediate (which is formed in several hundreds of picosecond time-scale after the blue light irradiation [2])by the TG method. The ∆H value of this intermediate was ~85 (±12) KJ/mole at 250C. This ∆H value decreased
with decreasing the temperature and this temperature dependence indicates nonzero heat capacity change
between the ground state and the intermediate state (∆Cp). Since the heat capacity is an indicator of the solvent
accessible surface area, this positive ∆Cp value may indicate that the hydrophobic surface area is exposed inthis intermediate.
2.5
3.0
2.5

ITrL\a.u.

ITG\a.u.

2.0
1.5
concentration

1.0

2.0
1.5

reference

1.0

0.5

(v)

AppA

0.5

0.0

0.0

0.00

0.01

0.02

t\s0.03

0.04

0.05

Fig.1: Concentration dependence of the TG
signals of AppA at q2=1.3×1012 m-2.

0.00.1t\s0.2

0.3

Fig.2: Typical TrL signals of calorimetricreference sample and AppA at 298K.

We have also measured the enthalpy change associated with the dimerization reaction by the TrL
method (Fig.2). Analysis of the TrL signal indicates that this dimer formation is accompanied by negative ∆H.
That means that dimerization reaction is accompanied by heat release rather than heat absorption. Thus weconclude that the dimerization reaction of AppA stabilize the product energetically. We hope that these
thermodynamic parameters may give new insight to the photocycle of the BLUF domain of AppA.

References:[1] S. Masuda, C. E. Bauer; Cell, 2002, 110, 613.
[2] M. Gauden, S. Yeremenko, W. Laan, I. H. van Stokkum, J. A. Ihalainen, R. van Grondelle, K. J. Hellingwerf,
J. T. Kennis; Biochemistry, 2005, 44, 3653.
[3] T. Eitoku, Y. Nakasone, D. Matsuoka, S. Tokutomi, M. Terazima; J. Am. Chem. Soc., 2005, 127, 13238.
[4] J. S. Khan, Y. Imamoto, M. Kataoka, F. Tokunaga, M. Terazima; J. Am. Chem. Soc., 2006, 128, 1002.
[5] S. Nishida, T. Nada, M. Terazima; Biophys. J., 2005, 89, 2004.
[6] W. Laan, M. A. van der Horst, I. H. van Stokkum, K. J. Hellingwerf; Photochem. Photobiol., 2003, 78, 290.