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J. Phys. Chem. B 2008, 112, 1494-1501
Energetics and Role of the Hydrophobic Interaction during Photoreaction of the BLUF Domain of AppA Partha Hazra,† Keiichi Inoue,† Wouter Laan,‡ Klaas J. Hellingwerf,‡ and Masahide Terazima*,† Department of Chemistry, Graduate School of Science, Kyoto UniVersity, Kyoto 606-8502, Japan, and Laboratory for Microbiology, Swammerdam Institute for Life Science, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands ReceiVed: August 22, 2007; In Final Form: NoVember 7, 2007
A recently developed method for time-resolved thermodynamic measurements was used to study the photochemical reaction(s) of the BLUF domain of AppA (AppA-BLUF), which has a dimeric form in the ground state, in terms of the energetics and heat capacity changes (∆Cp) in different time domains. The enthalpy change (∆H) of the first intermediate that forms within 1 ns after photoexcitation was 38 ((8) kJ mol-1 at 298 K. The heat capacity change (∆Cp) upon formation of this intermediate was positive [1.4 ((0.3) kJ mol-1 K-1]. This positive ∆Cp suggests that the hydrophobic surface area of AppA-BLUF exposed to the bulk solvent increased. After this initial transition, a dimerization reaction with another ground-state dimer (i.e., tetramer formation) takes place. Upon this reaction, the energy was stabilized to 26 ((6) kJ mol-1 at 298 K. Interestingly, the dimer formation was accompanied by a larger but negative ∆Cp [-6.0 ((1) kJ mol-1 K-1]. This negative ∆Cp might indicate buried hydrophobic residues at the interface of the dimer and/or the existence of trapped water at the interface. We suggest that hydrophobic interactions are the main driving force for the formation of the dimer upon photoactivation of AppA-BLUF.
1. Introduction The light- and redox-sensitive transcriptional antirepressor AppA was discovered in Rhodobacter sphaeroides, a proteobacterium that can grow both phototrophically in the light and chemotrophically in the dark, when oxygen or alternative electron acceptors are present in sufficient amounts. This signal receptor protein has at its N-terminus one of the most extensively characterized BLUF (sensor of blue light using FAD) domains,1-5 which serves as a light sensor that communicates the presence of blue light to a downstream output domain. When Rhodobacter cells are grown at low oxygen tensions, the dark-adapted form of AppA is able to bind to, and thereby inactivate, the repressor PpsR, thus allowing RNA polymerase to maximally transcribe photosynthesis genes.1-5 Blue light excites the FAD in the BLUF domain of AppA, which thereby is transformed into a signaling state that is incapable of interacting with the photosynthesis-gene repressor PpsR.1-5 Under these conditions, maximal repression of photosynthesis-gene expression occurs.1-5 The dark- (receptor-) state structure of the BLUF domain of AppA was resolved by X-ray diffraction analysis6,7 and NMR spectroscopy.8 Various techniques have been used to reveal the photochemistry and primary signaling processes of the BLUF domain of AppA (AppA-BLUF).9-16 Upon illumination, an intermediate, which exhibits a ∼10 nm red-shifted absorption spectrum, is formed (AppA-BLUFred) and decays back to the initial ground state with a rate of ∼1 × 10-3 s-1.9,10 Intramolecular electron-/ proton-transfer processes and subsequent alterations in the * To whom correspondence should be addressed. Phone: +81-75-7534026. Fax: +81-75-753-4000. E-mail:
[email protected]. † Kyoto University. ‡ Swammerdam Institute for Life Science.
hydrogen bonding between the flavin cofactor and amino acid side chains have been suggested to underlie this red shift.6,10-15 FTIR, time-resolved fluorescence, NMR, and steady-state Raman studies indicated that the BLUF domain of AppA undergoessomestructuralchangesuponblue-lightillumination.10-17 Ultrafast fluorescence, femtosecond transient absorption, and nanosecond flash photolysis spectroscopy on the BLUF domain of AppA revealed the transient formation of several intermediates with lifetimes ranging from the picosecond to the nanosecond time scale and showed that AppA-BLUFred is created within 1 ns.10,11 It was recently established that the ground state of the BLUF domain of AppA exists as a dimer even in very dilute solution.15 Although no further absorption change occurs after the formation of AppA-BLUFred, a time-resolved diffusion study showed that AppA-BLUFred subsequently associates with ground-state AppA-BLUF to form a dimer in the photoactivated state.18 [Because the BLUF domain of AppA exists as a dimeric form in the ground state, this dimerization reaction indicates formation of a tetramer. In this study, a BLUF domain of AppA corresponding to residues of 5-125 was used. This truncated AppA having the dimer form is referred to as AppA-BLUF hereafter in this work. Therefore, the dimer of AppA-BLUF, (AppA-BLUF)2, in this article denotes the tetramer of the BLUF domain of AppA.] However, despite these efforts, the photochemistry, structural dynamics, nature of the transients, and driving force behind this (AppA-BLUF)2 formation reaction in the photoactivated state of AppA-BLUF have not yet been resolved. To clarify the nature of the intermolecular interactions upon (AppA-BLUF)2 formation, we have studied the thermodynamic properties of the intermediate species that are formed in the photoactivation process of the BLUF domain. Of these, the heat capacity change (∆Cp) is particularly informative with respect
10.1021/jp0767314 CCC: $40.75 © 2008 American Chemical Society Published on Web 01/12/2008
Photoreaction of the BLUF Domain of AppA to altered protein-protein and protein-solvent interactions.19-29 Accordingly, the natures of folding and unfolding reactions have been extensively investigated from the viewpoint of ∆Cp.19-28 For example, a positive ∆Cp has been interpreted as a characteristic signature of protein unfolding, and likewise, a negative ∆Cp is considered to indicate protein folding.19-28 To elucidate how molecular recognition processes govern complex biological interactions, there is burgeoning interest in the measurement of the heat capacity changes to better understand the noncovalent forces that drive protein-protein interactions.28-33 For example, it was observed that protein-protein interactions, protein-DNA interactions, and protein-ligand interactions are accompanied by a large negative ∆Cps.28-33 The molecular origins of ∆Cp have been discussed previously as described in a later section.28-33 Differential scanning calorimetry and isothermal titration calorimetry are two main techniques that have been used for determinations of ∆Cp. However, both techniques are applicable solely to steady-state protein structures under equilibrium conditions. Knowledge of this property of transient intermediate species involved in biological processes has thus far been very limited, because of the lack of suitable experimental techniques. To alleviate this situation, the pulsed laser-induced transient grating (TG) and transient lens (TrL) methods have been developed and successfully used to detect spectrally silent dynamics with simultaneous quantitative measurement of some key thermodynamic parameters.34-38 The enthalpy change (∆H), partial molar volume change, and thermal expansion coefficient have been measured in various time domains.34-39 In this article, we describe a study of the nature of the transient intermediates that are formed during the photocycle of AppA-BLUF with respect to their thermodynamic characteristics, in particular, their enthalpy content and heat capacity, by the pulsed laser-induced TG and TrL methods. The heat capacity change (∆Cp) upon formation of this intermediate was positive [1.4 ((0.3) kJ mol-1 K-1]. On the other hand, interestingly, we found that ∆Cp for (AppA-BLUF)2 formation was largely negative. We discussed the molecular origin of these changes, and we suggest that the driving force of the dimerization reaction is the hydrophobic interactions that are induced by the conformational changes occurring upon light illumination. 2. Experimental Section Expression and Purification of the BLUF Domain of AppA. The BLUF domain of wild-type AppA (i.e., the 5-125 N-terminal amino acid residues) was expressed and purified essentially as described previously:4,18 Heterologous protein overproduction was performed in E. coli M15 (pREP4), grown in production broth (PB, which contains 20 g L-1 tryptone, 10 g L-1 yeast extract, 5 g L-1 dextrose, 5 g L-1 NaCl, and 8.7 g L-1 K2HPO4, pH 7.0). Ampicillin and kanamycin were used at 100 and 50 µg mL-1, respectively. Prior to purification, which was performed using a nickel-affinity resin, cell-free extract was incubated for 1 h on ice, with a large molar excess of FAD. Purified protein was dialyzed to 10 mM Tris-HCl, pH 8.0, and stored at -20 °C. The purity of the samples was checked by SDS-PAGE, using the PHAST System (Amersham Biosciences), and by UV/vis spectroscopy. The flavin composition of the purified protein was determined by thin-layer chromatography (TLC).4 TG and TrL Experiments. The experimental setup for the TG34-39 and TrL39-41 experiments was similar to that reported in our previous publications. A laser pulse from a dye laser (Lumomics, Ontario, Canada, HyperDye 300; λ ) 465 nm)
J. Phys. Chem. B, Vol. 112, No. 5, 2008 1495 pumped by an excimer laser (Lambda Physik, Go¨ttingen, Germany, XeCl operation; λ ) 308 nm) was split into two by a beam splitter, and the beams were crossed inside a quartz sample cell (optical path length ) 2 mm). The laser power of the excitation was
