시분해 형광을 통한 들뜬 상태 양성자 이동 분자와 형광 단백질의 분자 동역학 연구

Abstract

Progress in femtosecond spectroscopy has enlarged the window of observable dynamics. Many of molecular motions that we are interested in occur at ultrafast time scale of 10~100 fs, the period of molecular vibrations. Real time observation of structural evolution of molecules, reaction pathway and interaction between system and bath has been the goal of time-resolved spectroscopy. However, the temporal resolution and the adequate spectral range have been always obstacles. In this thesis, time-resolved fluorescence (TRF) spectroscopy is exploited as a powerful tool of molecular dynamics. Although TRF uses optical pulses to induce the change in population of electronic states, we can obtain not only the kinetic information but also the vibrational information of molecules in time through impulsive excitation of vibrations. Since the transient absorption signals are composed of ground state bleaching, stimulated emission from excited state and excited state absorption, time-resolved fluorescence (TRF) is suitable to detect the excited state dynamics exclusively.Another spectroscopic technique we will present in this thesis is Terahertz time-domain spectroscopy (THz TDS) which uses THz pulses to pump or probe a system. THz region is a new light source which promotes vibrational or rotational transition of molecules and it can provide femtosecond time-resolution. Therefore it is possible to detect the structural evolution of molecules in the excited state directly with the optical pump pulse. In the field of THz spectroscopy, it is important to enhance the S/N ratio of the spectroscopic system which immediately affects to the detection time (or number of scan) because the THz spectroscopy measures the electric field in time. Thus we utilize the cavity-dumped Ti:sapphire oscillator as an optical source of THz generation and detection due to the stability of the laser system. Generation and detection of THz pulses utilizing nonlinear crystal i.e., GaSe open the possibility of broad band (optical rectification) and spectrally resolved (DFG) detection system. This topic will be discussed specifically in chapter 2. The molecular reaction dynamics of the proton transfer has been the subject of extensive investigations, as it is one of the most important reactions in chemistry and biology. Excited state intramolecular proton transfer (ESIPT) affords a convenient molecular system of proton transfer that can be initiated by ultrashort optical pulses and interrogated subsequently by various time-resolved spectroscopic techniques. In chapter 3, we investigate the ESIPT dynamics of 2-(2’-Hydroxyphenyl)benzothiazole (HBT) and 10-Hydroxybenzo[h]quinoline (HBQ), which are the representative ESIPT molecules that show Stokes shifts as large as 10000 cm-1. The role of proton in the course of proton transfer, whether it is active or passive, has been the subject of intense investigations. We demonstrate the active role of proton in ESIPT of HBQ by directly resolving the ultrafast dynamics and the isotope dependence. The ESIPT of HBQ proceeds in 12±6 fs, and the rate is slowed down to 25±5 fs for DBQ where the reactive hydrogen is replaced by deuterium. The results are consistent with the ballistic proton wavepacket transfer within the experimental uncertainty. This ultrafast proton transfer leads to the coherent excitation of the vibrational modes of the product state. In contrast, ESIPT of HBT is much slower at 62 fs and shows no isotope dependence implying complete passive role of the proton. In chapter 4, molecular dynamics of green fluorescent protein (GFP) chromophore derivatives will be exploited by TRF spectroscopy. GFP has been widely used as biological markers in living cells, since it is noninvasive and does not need any substrate or coenzyme to fluoresce. The chromophore without protein matrix shows low quantum yield of fluorescence due to the conformational relaxation. To suppress this conformational relaxation, o-HBDI was synthesized as an ortho isomer of the fluorophore of GFP, 4-(4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (p-HBDI). o-HBDI has an intramolecular hydrogen bonding and undergoes ESIPT reaction with ultrafast reaction rate. Various derivatives of o-HBDI, which have different functional groups like –Br, -NO2, -OMe and –phenyl are synthesized to clarify the effect of the electronic property to ESIPT reaction. TRF spectroscopy and quantum mechanical calculations revealed tendencies in the largely modulated vibrational modes owing to the ultrafast ESIPT reaction. However the ESIPT reaction rate was irrespective of the electron withdrawing or electron donating capabilities. The excited state dynamics of various mutants of red fluorescent protein (RFP) was investigated with femtosecond TRF spectroscopy in chapter 5. Red shifted fluorescence of biological markers has been demanded due to the autofluorescence of the cell environment. Mutants from TagRFP to mKate absorb visible light about 550~580 nm and show small Stokes shift. TRF spectroscopy using OPO laser system provides visible pump and IR gate pulses. We obtained ultrafast solvation time about 200 fs in spite of the protein matrix, which is comparable to the bulk water solvation time. From QM/MM singlet point calculation of TagRFP, the interference of other electronic excited state can be eliminated. Therefore, the fast spectral shift about 200 fs can be assigned to a dynamic solvation alone. In addition, low frequency oscillation about 30 cm-1 was obtained for mKates and TagRFPs. This slow motion can be expected to a large skeletal motion related to cis-trans isomerization. From the study of structural analysis in steady state, most of chromophores of RFPs are cis (coplanar) in ground state except for TagRFP which has trans (coplanar) configuration.