Characterization of xanthorhodopsin from Salinibacterium xinjiangense and its selective binding of carotenoids
- 주제어 (키워드) Xanthorhodopsin , Salinibacterium xinjiangense , carotenoid
- 발행기관 서강대학교 일반대학원
- 지도교수 정광환
- 발행년도 2025
- 학위수여년월 2025. 2
- 학위명 석사
- 학과 및 전공 일반대학원 생명과학과
- 실제 URI http://www.dcollection.net/handler/sogang/000000079440
- UCI I804:11029-000000079440
- 본문언어 영어
- 저작권 서강대학교 논문은 저작권 보호를 받습니다.
초록 (요약문)
Rhodopsin is a seven-transmembrane protein covalently linked with the retinal chromophore. It plays various roles through light stimulation and is found in all domains of life. Microbial rhodopsins are activated by light and can perform various functions, including proton and ion translocation, photo-sensory activities, and photo-enzymatic processes, many aspects of which are still awaiting exploration. This study investigated and characterized the microbial rhodopsin isolated from Salinibacterium xinjiangense (Sx), the cold-adapted microorganism, and was abbreviated as SxRho. SxRho was expressed and purified in E.coli. The purified SxRho in 0.02% DDM exhibited a maximum absorption at 530 nm at pH 7.0. Functional characterization of SxRho including proton pumping, pKa value, and protein kinetics were determined in both wild-type and mutants. SxRho is identified as a novel microbial rhodopsin belonging to the xanthorhodopsin type, which is phylogenetically closer to dual chromophores binding rhodopsin to bacteriorhodopsin (BR) or proteorhodopsin (PR). SxRho conserved Gly156, which is an essential residue for carotenoid binding in xanthorhodopsin. To investigate the carotenoid-binding properties, carotenoids were extracted from native Sx cells. Interaction studies between SxRho and carotenoids were conducted to compare native carotenoids with their analogs. The results indicate that SxRho selectively binds to the carotenoid produced by Sx, supporting the theory of a specific rhodopsin-carotenoid pairing. This binding was shown to protect SxRho under heat stress, suggesting a protective role for carotenoids in high-temperature environments.
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CHAPTER I: INTRODUCTION 8
1.1. Diversity of microbial rhodopsin 8
1.2. The first discovery of xanthorhodopsin 9
1.3. Xanthorhodopsin in Salinibacterium xinjiangense 10
CHAPTER II: MATERIALS AND METHODS 15
2.1. Molecular cloning and site-directed mutagenesis 15
2.2. Protein expression and purification 16
2.3. Absorption spectroscopy and pKa measurement 17
2.4. Light-induced proton pumping measurements 18
2.5. Light-induced differential spectrum experiment 19
2.6. Carotenoid interaction measurements 19
2.7. Protein stability measurement under thermal stress 21
CHAPTER III: RESULTS AND DISCUSSION 22
3.1. Characterization of xanthorhodopsin from Salinibacterium xinjiangense (SxRho) 22
3.1.1. UV-visible absorption of wild-type SxRho and mutants 22
3.1.2. pH titration and pKa measurement of SxRho wild-type and mutants 24
3.1.3. Proton pumping activity 29
3.1.4. Light-induced differential spectra 32
3.2. In vitro and in vivo study of the selective carotenoid binding 35
3.2.1. Isolation of carotenoid from Salinibacterium xinjiangense (Sx) and comparison with other carotenoids 35
3.2.2. In vitro and in vivo study of the selective binding of carotenoids 42
3.2.3. The protective function of carotenoid in SxRho against heat stress 45
CHAPTER IV: CONCLUSION 49
CHAPTER V: REFERENCE 50
