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FRET studies on TMR-labeled cytochrome c using bulk and single molecule spectroscopy to characterize the conformational heterogeneity

FRET studies on TMR-labeled cytochrome c using bulk and single molecule spectroscopy to characterize the conformational heterogeneity
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대학원 분자생명과학부
이화여자대학교 대학원
Local structures and conformational distributions of biomolecules are important in understanding their biological roles and fluorescence resonance energy transfer (FRET) is the spectroscopic ruler to measure the intramolecular distance of biomolecular system in the 10-100Å´ ranges. We studied the FRET of TMR labeled cytochrome c model systems in aqueous solution from ensemble measurements and at the single molecule level to investigate the conformational heterogeneity of protein. For this study, we labeled the thiolate sulfur of Cys102 at C-terminal in yeast cytochrome c (cyt c) with a tetramethylrhodamine (TMR) dye. The appreciable donor-acceptor fluorescence energy transfer was observed, due to spectral overlap of the heme absorption of the cyt c and the emission of TMR. The FRET efficiencies and the distances between the donor TMR and the acceptor heme group were obtained from time-resolved fluorescence lifetime measurements. The fluorescence of the labeled TMR was significantly quenched by energy transfer to the heme in the native conformation, but fluoresced intensely and the lifetime increased when the protein is unfolded in guanidine hydrochloride (GuHCl) solution. The flexibility of the donor TMR and the distance between the donor TMR and the acceptor heme group were optimized by inserting an inert saturated hydrocarbon linker and appreciable donor-acceptor FRET was observed. By employing the fluorescence anisotropy measurement, the relative orientation between two chromophores in cyt c was obtained under various conditions. We also studied FRET phenomena using single molecule fluorescence spectroscopy. Single molecule study was investigated by employing a home-built time-resolved confocal fluorescence microscope. The donor-acceptor distance distributions were obtained from the fluorescence lifetime data of single cyt c molecules in buffer solution and in sol-gel (hydrogel) matrices and conformation mapping of native and denatured cyt c proteins was assessed in the function of the denaturant, guanidine hydrochloride (GuHCl) concentration. The lifetime distribution obtained by single molecule measurements was continuous and had a relatively wide range compared with the data from the bulk measurements. ;생물질의 국부적 구조와 형상 분포는 생물학적 역할을 규명하는 데 있어 매우 중요하며, fluorescence resonance energy transfer (FRET)은 생분자 시스템에서 분자내의 10-100Å´ 범위의 거리를 측정하는 데 있어 널리 사용되어 오고 있는 유용한 spectroscopic ruler라 할 수 있다. 본 연구는 TMR을 labeling한 cytochrome c 단백질을 이용해 수용액상태에서의 ensemble 측정과 단분자 수준에서의 측정을 토대로 하여 단백질의 conformational heterogeneity를 살펴보았다. 이를 위해 yeast cytochrome c (cyt c)를 model system으로 하여 cyt c의 C-terminal의 102 cysteine residue의 thiolate sulfur 에 tetramethylrhodamine (TMR) dye를 labeling하였다. 그 결과 cyt c의 heme group을 acceptor로, labeling한 dye는 donor로 하는 FRET(Fluorescence Resonance Energy Transfer) system이 구축되었고, 이들 donor-acceptor 간의 fluorescence energy transfer가 일어남을 관찰하였다. 이는 acceptor인 cyt c의 heme group의 absorption과 donor인 labeled TMR의 emission 간의 spectral overlap에 의한 것이다. 시분해 형광수명 측정을 통해 FRET efficiencies와 donor TMR와 acceptor heme group 간의 거리를 계산하였다. Cyt c 단백질의 native conformation에서는 heme group으로의 에너지 전달로 인해 labeling된 TMR의 형광이 크게 감소하였으나, denaturant인 guanidine hydrochloride (GuHCl)의 첨가로 단백질 구조가 unfolded된 상태에서 형광은 다시 증가하였다. Inert saturated hydrocarbon linker를 삽입하여 donor TMR의 flexibility와 donor TMR과 acceptor heme group간의 거리를 변화시켰으며 이 경우에도 donor와 acceptor 간의 FRET 현상이 일어남이 관측되었다. 또한 형광 비등방성 측정 실험을 통해 cyt c에 labeling된 TMR과 heme group 두 chromophores 간의 상대적인 배향에 대해 연구하였다. 또한 단분자 형광 분광학을 이용해 FRET 현상을 연구하였는데 단분자 연구는 home-built time-resolved confocal fluorescence microscope를 이용하여 진행되었다. Buffer solution과 sol-gel (hydrogel) matrices 내의 single cyt c molecules의 형광 수명을 측정함으로써 donor TMR과 acceptor heme group 간의 거리 분포를 얻었으며 이를 토대로, denaturant인 guanidine hydrochloride (GuHCl)의 농도를 증가시키며 native 상태와 denatured된 상태의 cyt c 단백질의 형상 분포를 mapping할 수 있었다. 이러한 단분자 측정 결과는 bulk 측정 결과에 비해 연속적이며 상대적으로 더 넓은 범위의 분포를 나타내었다.
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