Yusuke Nakasone

Time-Resolved Tracking of Interprotein Signal Transduction: Synechocystis PixD–PixE Complex as a Sensor of Light Intensity

PixD (Slr1694) is a blue light receptor that contains a BLUF (blue light sensors using a flavin chromophore) domain. A protein-protein interaction between PixD and a response regulator PixE (Slr1693) is essential to achieve light signal transduction for phototaxis of the species. Although the initial photochemical reaction of PixD, the red shift of the flavin absorption spectrum, has been investigated, the subsequent reaction dynamics remain largely unresolved. Only the disassembly of the PixD(10)-PixE(5) dark complex has been characterized by static size exclusion chromatography. In this report, interprotein reaction dynamics were examined using time-resolved transient grating spectroscopy. The dissociation process was clearly observed as the light-induced diffusion coefficient change in the time domain, and the kinetics was determined. More strikingly, disassembly was found to take place only after photoactivation of two PixD subunits in the complex. This result suggests that the biological response of PixD does not follow a linear correlation with the light intensity but appears to be light-intensity-dependent.

Temperature-Sensitive Reaction of a Photosensor Protein YcgF: Possibility of a Role of Temperature Sensor

The spectrally silent photoreaction of a blue light sensor protein YcgF, composed of the N-terminal BLUF domain and the C-terminal EAL domain, was investigated by the time-resolved transient grating method. Comparing photoinduced reactions of full-length YcgF with that of the BLUF-linker construct, it was found that a major conformation change after photoinduced dimerization is predominantly localized on the EAL domain. Furthermore, the photoinduced conformational change displayed significant temperature dependence. This result is explained by an equilibrium of reactive and nonreactive YcgF species, with the population of photoreactive species decreasing as the temperature is lowered in the dark state. We consider that the dimer form is the nonreactive species and it is the dominant species at lower temperatures. The temperature sensitivity of the photoreaction of YcgF suggests that this protein could have a biological function as a temperature sensor as well as behaving as a light sensor.

Light-induced conformational change and transient dissociation reaction of the BLUF photoreceptor Synechocystis PixD (Slr1694).

The light-induced reaction of the BLUF (blue light photoreceptor using flavin adenine dinucleotide) photoreceptor PixD from Synechocystis sp. PCC6803 (Slr1694) was investigated using the time-resolved transient grating method. A conformational change coupled with a volume contraction of 13 mL mol(-1) was observed with a time constant of 45 ms following photoexcitation. At a weak excitation light intensity, there were no further changes in volume and diffusion coefficient (D). The determined D-value (3.7×10(-11) m(2) s(-1)) suggests that PixD exists as a decamer in solution, and this oligomeric state was confirmed by size-exclusion chromatography and blue native polyacrylamide gel electrophoresis. Surprisingly, by increasing the excitation laser power, we observed a large increase in D with a time constant of 350 ms following the volume contraction reaction. The D-value of this photoproduct species (7.5×10(-11) m(2) s(-1)) is close to that of the PixD dimer. Combined with transient grating and size-exclusion chromatography measurements under light-illuminated conditions, the light-induced increase in D was attributed to a transient dissociation reaction of the PixD decamer to a dimer. For the M93A-mutated PixD, no volume or D-change was observed. Furthermore, we showed that the M93A mutant did not form the decamer but only the dimer in the dark state. These results indicate that the formation of the decamer and the conformational change around the Met residue are important factors that control the regulation of the downstream signal transduction by the PixD photoreceptor.

Stability of Dimer and Domain-Domain Interaction of Arabidopsis Phototropin 1 LOV2

Transient grating signals after photoexcitation of Arabidopsis phototropin 1 light-oxygen-voltage 2 (phot1LOV2) domain without the linker were found to be very sensitive to temperature. In particular, the diffusion signal drastically increased with rising temperature. The signal was consistently explained by the superposition of the photo-induced dissociation and association reactions. This observation indicated the presence of an equilibrium between the monomer and dimer forms of the phot1LOV2 domain in the dark. The equilibrium was confirmed by a gel chromatographic technique. The equilibrium constants at various temperatures were calculated from the fraction of the dimer, and the stabilization enthalpy and entropy were determined. Interestingly, the transient grating signal of phot1LOV2 with the linker (phot1LOV2-linker), which exists as the monomer form, was also temperature dependent; the diffusion signal intensity decreased with increasing temperature. Because the diffusion signal reflects a conformation change of the linker upon photoexcitation, this temperature dependence indicated that there were two forms of the phot1LOV2-linker. One form exhibited a conformational change upon photoexcitation whereas the other form showed no change. These two forms are not distinguishable spectroscopically. The fraction of these species depended on the temperature. Considering the monomer-dimer equilibrium of the phot1LOV2 domain, we suggest that the nonreactive form possesses the linker region that is dissociated from the LOV2 domain. Because the dissociation of the linker region from the LOV2 domain is a key step for the conformation change of the phot1LOV2-linker to induce biological activity, we proposed that the phototropins could have a role as a temperature sensor.

On the binding of BODIPY-GTP by the photosensory protein YtvA from the common soil bacterium Bacillus subtilis

The YtvA protein, which is one of the proteins that comprises the network carrying out the signal transfer inducing the general stress response in Bacillus subtilis, is composed of an N‐terminal LOV domain (that binds a flavin [FMN]) and a C‐terminal STAS domain. This latter domain shows sequence features typical for a nucleotide (NTP) binding protein. It has been proposed (FEBS Lett., 580 [2006], 3818) that BODIPY‐GTP can be used as a reporter for nucleotide binding to this site and that activation of the LOV domain by blue light is reflected in an alteration of the BODIPY‐GTP fluorescence. Here we confirm that BODIPY‐GTP indeed binds to YtvA, but rather nonspecifically, and not limited to the STAS domain. Blue‐light modulation of fluorescence emission of YtvA‐bound BODIPY‐GTP is observed both in the full‐length YtvA protein and in a truncated protein composed of the LOV‐domain plus the LOV‐STAS linker region (YtvA1–147) as a light‐induced decrease in fluorescence emission. The isolated LOV domain (i.e. without the linker region) does not show such BODIPY‐GTP fluorescence changes. Dialysis experiments have confirmed the blue‐light‐induced release of BODIPY‐GTP from YtvA.

A way to sense light intensity: Multiple-excitation of the BLUF photoreceptor TePixD suppresses conformational change

TePixD, a cyanobacterial sensor of blue light using flavin adenine dinucleotide (FAD) (BLUF) which exists in a decamer form, was found to exhibit photoreaction sensitive to light intensity. While the number of excited molecules increased monotonically as the laser power increased, the number of decamers exhibiting a global conformational change initially increased, and then decreased with the increase of excitation intensity. This unusual power dependence was analyzed based on a Poisson distribution equation, demonstrating that decamers containing more than one excited monomer subunit do not undergo conformational change. Our results suggest that TePixD functions not only as a photosensor, but also by sensing light intensity.

Modeling the functioning of YtvA in the general stress response in Bacillus subtilis

The blue-light photoreceptor YtvA activates the general stress response (GSR) of Bacillus subtilis by activating a large protein complex (the stressosome). We have constructed a model for the YtvAs photocycle, and derived an equation for the fraction of YtvA in the light-induced signaling state at a given light intensity. The model was verified experimentally in vitro on wild type YtvA and on an R63K mutant with faster recovery kinetics. Application of the model to the activation of the GSR at various light intensities in vivo revealed that the GSR is more sensitive to light than would be expected based on YtvAs in vitro kinetics. These results were confirmed with the R63K mutant and a slower-recovering V28I mutant. Additionally, we have demonstrated the presence of a near-UV-light-induced branching reaction that converts the signaling state of YtvA to the dark state. Extension of the model with this reaction shows that it does not contribute significantly to the in vivo blue-light response. The model represents an important step towards a complete systems biology model of the GSR.

Kinetics of Conformational Changes of the FKF1-LOV Domain upon Photoexcitation

The photochemical reaction dynamics of a light-oxygen-voltage (LOV) domain from the blue light sensor protein, FKF1 (flavin-binding Kelch repeat F-box) was studied by means of the pulsed laser-induced transient grating method. The observed absorption spectral changes upon photoexcitation were similar to the spectral changes observed for typical LOV domain proteins (e.g., phototropins). The adduct formation took place with a time constant of 6 μs. After this reaction, a significant conformational change with a time constant of 6 ms was observed as a change in the diffusion coefficient. An FKF1-LOV mutant without the conserved loop connecting helices E and F, which is present only in the FKF1/LOV Kelch protein 2/ZEITLUPE family, did not show these slow phase dynamics. This result indicates that the conformational change in the loop region represents a major change in the FKF1-LOV photoreaction.

When is the helix conformation restored after the reverse reaction of phototropin

Following the disruption of the covalent bond between the cysteine and flavin of Phot1LOV2-linker, the unfolded conformation of the linker folds with a time constant of 13 ms, which is considerably (approximately 10(4) times) slower than the helix formation rate measured for an alpha-helical polypeptide in solution.