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.

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