Study of conformational dynamics of LOV2 and PYP photoreceptor proteins using transient grating spectroscopy and transient absorption spectroscopy = 과도 격자 분광법과 과도 흡수 분광법을 통한 LOV2와 PYP 광수용체 단백질의 구조 동역학에 대한 연구

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Plants and several organisms use light energy and converts them into another form of energy, for performing several functions. Therefore, in order to regulate the functions in response to light by plants and organisms, so-called “photoreceptors” (proteins) are used. Phototropins are photoreceptors, which performs the function of Phototropism in addition to the functions like stomatal opening, chloroplast movement in higher plants. Phototropins responds to blue-light and exist, as isoforms (Phototropin 1 and Phototropin 2) in higher plants.LOV2 domain from Phot 1, is one such example that responds to blue light and involves in activation of kinase domain. Similarly, PYP like photoreceptors of also responds to blue-light and involves in the negative phototaxis of E.halophila. Number of domains together constitute a photoreceptor protein, which responds to light and controls the function by interacting with each other. LOV2 domain interacts with LOV1 domain and tranduces the signal to the kinase domain. PYP on the other hand, is a small protein that stands as a prototype for the PAS domains (which has multidomain architecture) and involves in signal transduction in response to changes with the environment and to changes within the cell. LOV2 domain switches from dark state to light adapted adduct state (Flavin mononucleotide (FMN)-cysteinyl adduct). Upon irradiation with blue light, pCA (p-coumaric acid-cofactor), which is a chromophore of PYP, undergoes photoisomerisation.. These kind of photoreceptors are in the lime light of research, since, the conformational changes of these photoreceptors when “controlled by light”, would pave the way for production of “optogenetic tools”. The structural changes associated conformation dynamics of As LOV2-J$\alpha$ and As LOV2-$\Delta$J$\alpha$ domains were studied using Transient Grating (TG) and Transient Absorption (TA) spectroscopies. Based on the fitting results of decay profiles monitored at 660nm we found that FMN-Cysteinyl adduct formation in As LOV2-J$\alpha$ and As LOV2-$\Delta$J$\alpha$ domains occurs with time constants of 2.2 and 2.1 $\mu$s, respectively. The dark recovery rate from the fitting result was 110s for both domains, which indicates that the J$\alpha$ truncation did not alter the recovery rate. The changes in Diffusion coefficient was measured using TG spectroscopy to observe the conformational changes (spectrally silent features) that occurs after the adduct formation. Based on the D value, As LOV2-J$\alpha$ and As LOV2-$\Delta$J$\alpha$ domains exist as monomer and dimer in the dark state respectively. The As LOV2-J$\alpha$ domain undergoes photoinduced dimerization (1.2ms), by the photoinduced association of ground state domain with photoinduced domain, which then follows with J$\alpha$ helix unfolding. The As LOV2-$\Delta$J$\alpha$ forms monomer (0.98ms) upon photoinduction proceeding with minor structural arrangements within the core. Therefore, the J$\alpha$ helix controls the equilibrium shift between monomer and dimer of the LOV2 domain. On the other hand, the effect of N-terminal extension (by length and sequence) on the photocycle of PYP studied using Transient absorption spectroscopy showed that, the Dark recovery (pB2to pG) was accelerated when compared to wild-type PYP,by extending the length (5G-PYP, 15G-PYP, 20G-PYP) of N-terminal. Only transient state that is being altered by extending N-terminal of PYP is pB2topG. Therefore, the lifetime of the transient signaling state (pB2topG) could be regulated by extending the N-terminal.
Ihee, Hyotcherlresearcher이효철researcher
Description
한국과학기술원 :화학과,
Publisher
한국과학기술원
Issue Date
2019
Identifier
325007
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 화학과, 2019.2,[v, 43 p. :]

Keywords

Phototropin▼aphotoactive yellow protein▼aoptogenetic tool▼atransient grating▼atransient absorption; 포토트로핀▼a비롯된 광활성 황단백질▼a광유전학적 도구▼a과도 격자▼a과도 흡광

URI
http://hdl.handle.net/10203/265493