Studies of the biomolecular dynamics using the laser-induced transient grating method

Abstract

Most proteins carry out their biological functions through the conformational change induced by various internal or external stimuli. In addition, the conformational change of a protein have an important role in the signal transduction process in numerous biological systems. In many biological systems, the photo-induced conformational change of a protein generate various biological signal, which is transduced to the signaling partner through the molecular interaction. Hence, the conformational dynamics of many photoactive proteins is an important key to understanding the functional mechanism of the protein. Thus, the photodynamics of many photoactive proteins have been extensively studied using various time-resolved spectroscopic techniques. In this thesis, I studied the photo-induced conformational change of hemoglobin (Hb), cytochrome c (Cytc) and photoactive yellow protein (PYP) using laser-induced transient grating (TG) spectroscopy. Generally, the TG signal after photoexcitation of a protein contains the information of the refractive index changes occurring during the reaction. In addition, the diffusion process of a chemical species involved in photoreaction can detected by using TG spectroscopy and consequently the diffusion coefficients of chemical species can be determined. Therefore, the TG signal provides the information on the global conformational change of the protein from the change in the diffusion coefficient of a protein. On one hand, the transient absorption (TA) spectroscopy is also utilized to obtain the local conformational change nearby the chromophore. First, the folding dynamics of Cytc initiated by the photodissociation of the CO ligand was investigated by using TG and TA spectroscopies. After the photodissociation of CO ligand from Cytc-CO in strongly basic condition, the TG signals show a q-dependence, reflecting time-dependent protein diffusion coefficient for the protein folding kinetics. The folding kinetics of Cytc shows a fast conformational change leading to the volume expansion ($1.4 \times 10^{6} s^{-1}$) and the tertiary structural change of a protein accompanying the diffusion coefficient change with $256 \pm 5 s^{-1}$. The optically triggered folding reaction of Cytc observed as the change of the diffusion coefficient supports two-state folding kinetics rather than multi-state folding kinetics (sequential mechanism). In addition, the measured diffusion coefficient for the unfolded state provides strong evidence that the unfolded state at pH 13 is partially unfolded unlike that induced by a denaturant. Second, by measuring the TG signal of Cytc-CO in the presence of a denaturant, it was clearly detected the change of diffusion coefficient that reflects the size change of Cytc upon photodissociation of the CO ligand from unfolded Cytc-CO. The quantitative analysis of TG signals supports that the optically triggered folding reaction of Cytc in the presence of a denaturant takes place through a detectable intermediate (three-state folding kinetics). This is in contrast with the two-state folding dynamics of Cytc under a denaturant-free environment without any detectable intermediate. From the quantitative global analysis of the TG signals, the rate constants for the U $\rightarrow$ I and I $\rightarrow$ N transitions in a CAPS buffer solution (pH 7) at room temperature in the presence of a denaturant at various concentrations are determined to be $1065 \pm 17$ to $3476 \pm 10^{3} s^{-1}$ and $101 \pm 6$ to $589 \pm 21 s^{-1}$, respectively. In addition, the activation energies ($E_a$) for the U $\rightarrow$ I and I $\rightarrow$ N transitions are determined to be $8.7 \pm 1.0 kcal/mol$ and $7.1 \pm 1.3 kcal/mol$, respectively. The folding dynamics of Cytc initiated by the CO photolysis is discussed based in terms of the protein size change. Third, the quaternary structural transition between the R and T states of Hb was investigated using the TA and TG spectroscopies. The result demonstrated that the dynamics at $~2 \mu$s observed in the TG experiment is due to the volume change caused by the quaternary structural change of Hb. This indicates that the dynamics at $~2 \mu$s corresponds to the fast step of the R？T transition, leading to a major quaternary structural change of Hb. Fourth, the effect of the elongated N-terminus on the photocycle of PYP has investigated using various spectroscopic techniques, such as circular dichroism (CD), nuclear magnetic resonance (NMR), TA and TG techniques. Upon the elongation of the N-terminus of PYP, the dark recovery time from $pB_{2}$ to pG becomes short, indicating that the pB state formed during the photocycle of PYP is greatly influenced by the elongation of N-terminus. This result is in contrast to those of the previous studies on the N-terminal truncated mutants of PYP, which showed the significantly slow dark recovery time compared to wild-type PYP. The acceleration of the dark recovery time of PYP with an elongated N-terminus is probably due to the interaction between extra N-terminal residues and the $\beta$-sheet of PYP. The results presented in this investigation clearly show that the $pB_{2}$ state, which is considered as a signaling state of PYP, can be regulated by controlling the length of N-terminal residues of PYP. Finally, the photodynamics of PYP in cell-mimetic environments was investigated. Biomolecules such as nucleic acids and proteins evolve and function within the crowded intracellular environments that are including a number of other biomolecules. In addition, the intracellular environments are very heterogeneous compared to in vitro experimental conditions in terms of temperature, pH, viscosity, etc. Consequently, this difference between in vivo and in vitro may result in the significant change in the structure and stability of biomolecules. To create such a crowded environment in solution, polyethylene glycols (PEG) with various molecular weights were used as the crowding agent in the investigation. As a result, we revealed that the kinetics related with blue-shifted intermediates, $pB_{1}$ and $pB_{2}$, were slowed down by the crowding agents. Consequently, the TA and TG result indicates that the kinetics of the pB states are affected by not only the change of the solution properties but also the conformation and size of the crowding agent.