Koji Okajima

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.

Transient conformational fluctuation of TePixD during a reaction.

Significance The role of conformational fluctuations in protein reactions has been frequently mentioned to discuss the reaction mechanism. Supporting evidence for the importance of the fluctuation has been reported by showing the relationship between the flexibility of the reactant structure and reaction efficiency. However, there has been no direct evidence showing that the fluctuation is indeed enhanced during the reaction, although recent molecular dynamic simulations pointed out the importance. Here, we focused our attention on the experimental proof of enhancement by the time-resolved transient grating method, which is a unique and powerful method. Our results showed that fluctuation is a key to understanding why light-stimulated proteins can transfer the signal without changing the averaged conformation. Knowledge of the dynamical behavior of proteins, and in particular their conformational fluctuations, is essential to understanding the mechanisms underlying their reactions. Here, transient enhancement of the isothermal partial molar compressibility, which is directly related to the conformational fluctuation, during a chemical reaction of a blue light sensor protein from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) was investigated in a time-resolved manner. The UV-Vis absorption spectrum of TePixD did not change with the application of high pressure. Conversely, the transient grating signal intensities representing the volume change depended significantly on the pressure. This result implies that the compressibility changes during the reaction. From the pressure dependence of the amplitude, the compressibility change of two short-lived intermediate (I1 and I2) states were determined to be +(5.6 ± 0.6) × 10−2 cm3⋅mol−1⋅MPa−1 for I1 and +(6.6 ± 0.7)×10−2 cm3⋅mol−1⋅MPa−1 for I2. This result showed that the structural fluctuation of intermediates was enhanced during the reaction. To clarify the relationship between the fluctuation and the reaction, the compressibility of multiply excited TePixD was investigated. The isothermal compressibility of I1 and I2 intermediates of TePixD showed a monotonic decrease with increasing excitation laser power, and this tendency correlated with the reactivity of the protein. This result indicates that the TePixD decamer cannot react when its structural fluctuation is small. We concluded that the enhanced compressibility is an important factor for triggering the reaction of TePixD. To our knowledge, this is the first report showing enhanced fluctuations of intermediate species during a protein reaction, supporting the importance of fluctuations.

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.

Two Intermediate States I and J Trapped at Low Temperature in the Photocycles of two BLUF Domain Proteins of Cyanobacteria Synechocystis sp. PCC6803 and Thermosynechococcus elongatus BP-1†

We identified the two intermediate states, I and J, that are common in the photocycles of the cyanobacterial BLUF (sensor of Blue Light Using Flavin) domain proteins of Slr1694 of Synechocystis sp. PCC6803 and Tll0078 of Thermosynechococcus elongatus BP‐1 by analyzing the absorption spectra at 5u2003K. Illumination at 5u2003K accumulated intermediate forms (designated as I5 and I9), which showed 5u2003and 9u2003nm redshifts of the absorption bands of flavin in the Tll0078 and Slr1694 proteins, respectively. I5 (I9) was converted into the next intermediate, which have 11u2003nm (14u2003nm) red‐shifted absorption bands J11 (J14) after dark annealing at 230u2003K (240u2003K). Further dark annealing at 280u2003K (270u2003K) of J11 (J14) produced the signal‐transmitting final form F490 (F495), with a small increase in the absorption at around 490u2003nm (495u2003nm). The results indicate that the BLUF proteins of Tll0078 and Slr1694 exhibit the common photocycle of D471 (D467) →I5 (I9) →J11 (J14) →F490 (F495) at low temperature. The transition temperatures for these intermediate forms differ for two proteins. The amount of I5 (I9) accumulated at 5u2003K was small and increased at a higher temperature, suggesting heterogeneity of the protein structure that determines the reaction pathway.

Macromolecular Crowding Effects on Reactions of TePixD (Tll0078)

To reveal macromolecular crowding effects on a chemical reaction of a BLUF (sensors of blue light using FAD) protein (PixD from a thermophilic cyanobacterium Thermosynechococcus elongatus BP‐1 [TePixD, Tll0078]), the photoreaction was studied at various concentrations of the macromolecule Ficoll‐70 by UV/Vis absorption spectroscopy and the pulsed laser‐induced transient grating (TG) method. The absorption spectrum did not change with varying concentration of Ficoll‐70. The crowding did not affect the quantum yield of the spectral red shift reaction, recovery rate of the product, rate constant of the volume change reaction and the magnitude of the volume change. However, the magnitude of the TG signal representing the diffusion‐sensitive conformation change significantly increased on addition of Ficoll‐70. This dependence was attributed to the crowding effect on the TePixD decamer–pentamer equilibrium in the solution. This result indicates that the TePixD reaction is more efficient in cellular than in in vitro conditions.

Photoreaction of Cyanobacterial BLUF Protein PixD Studied by Low Temperature Spectroscopy and Site-Directed Mutagenesis

Bluf Domain (Sensor Of Blue Light Using Fad) Is A Newly Identified Class Of Flavinbinding Photoreceptor Domain. Cyanobacterial Bluf-Domain Protein Pixd Has Been Revealed To Regulate The Positive Phototaxis Of Cells In The Cyanobacterium Synechocystis Sp. Pcc6803. Bluf Proteins Show 10–20 Nm Red-Shifts Of Their Flavin Absorption Bands Upon The Blue-Light Absorption. In This Study, We Studied The Photoconversion Mechanisms Of Tepixd Protein Of The Thermophilic Cyanobacterium Thermosynechococcus Elongatus Bp-1 By Low Temperature Spectroscopy And Sitedirected Mutagenesis. Although The Deletion Of The Tyr8 Residue Of Pixd Protein Had Been Shown To Abolish The Photoconversion At Room Temperature, We Show Here That Illumination At 80 K Produces A Normal Red-Shift Even In Y8F And Y8A Mutant Proteins. The Red-Shifted Forms That Are Trapped At 80 Kare Stable In The Dark, And Decay By Dark Annealing At 280 K. The Illumination At 150 K Of Y8F And Y8A Mutant Proteins, On The Other Hand, Formed An Anionic Form Of Flavin Suggesting The Existence Of Multiple Reaction Pathways. The Accumulations Of The Red-Shifted Forms In Y8F And Y8A Mutant Proteins At 80 K Occurred With 43- And 137-Times Slower Than That Of Wildtype Protein, Respectively, With The Extents At About 1/2 And 1/4 Of That Of Wild-Type. It Is Shown That The Photoconversion Of The Bluf-Domain Protein Occurs Even Without The Tyr8 Residue, And That Tyr8 Is Necessary To Enhance The Photoconversion Efficiency.