NMR 분광학을 이용한 막단백질 및 식물 대사체 연구

다국어 초록 (Multilingual Abstract)

Part. Ⅰ
Human transmembrane proteins (hTMPs) comprise a large proportion of integral membrane protein and perform many functions such as signal transduction, intercellular communication, enzyme and so on. To understand the function of hTMPs, it is important to identify their three-dimensional structure. But, since hTMPs are mostly composed of hydrophobic amino acids, there are difficulties in expression and purification of proteins. Therefore, we have optimized the expression and purification process and studied the structural analysis of syndecan-4 receptor (Syd4), human melanocortin-4 receptor (hMC4R), which are the types of hTMPs.
Syndecan 4 is a member of the syndecan family of heparan sulfate proteoglycans (HSPGs), consisting of a core protein and heparan sulfate. It is expressed in almost all cell types of mammals and performs various functions in vivo, such as cell-to-cell interaction, extracellular matrix interaction, growth-factor-receptor-activation, matrix adhesion, tumor suppression, and cell proliferation. The transmembrane region is expected to play an important role in this function. Therefore, experiments were conducted to determine the structure of syndecan 4 in the cell-like membrane. Syd4 with a few ecto-, transmembrane and cyto-domains was called syd4-eTC.
hMC4R is a G-protein coupled receptor (GPCR) that acts as a receptor for hormones that maintain energy homeostasis and transmits signals related to appetite. When hMC4R binds to α-MSH (α-Melanocyte stimulating hormone), it suppresses appetite, and when it binds to the antagonist Aguti related protein, AgRP, it enhances appetite. If aspartic acid, the 90th amino acid of hMC4R located second transmembrane (TM2), is replaced by asparagine, it is known that it can cause obesity due to inability to control appetite. We first optimized expression and purification process of wild type hMC4R-TM2 (wt-hMC4R-TM2) to see how the structures of wild type and mutant differ. We demonstrated methods that optimized for expression and purification process for each proteins and characterized them using various analytical methods such as mass spectrometry (MS), circular dichroism spectroscopy (CD), and NMR spectroscopy.

Part. Ⅱ
A chemical accident refers to any situation in which chemicals are leaked to people or the environment due to worker’s negligence, facility defects, obsolescence, natural disasters, transportation accident, and so on. Chemical accidents cause a variety of damages, including human health, ecosystems, material damage. Although objective data are needed to determine the extent of damage by chemical accidents, there is no specific method or procedure in our country for estimating the amount of damage for plant damage after a chemical accident. Therefore, it is necessary to establish a method and procedure for objectively determining the degree of damage to plants in the field of chemical accidents.
In this study, we optimized the pretreatment method of plant samples through literature review. Two experiments were conducted using standardized sample pretreatment methods. First, experiments were conducted in which the oak leaves exposed to artificial chemicals were compared to the normal oak leaves. Solution-State NMR, HR-MAS NMR and Solid-State NMR were used to observe changes in metabolites due to chemical compound. Second, experiments were conducted to compare normal lettuce with lettuce grown in chemically contaminated soil. Solution-State NMR and Solid-State NMR were used for metabolite analysis.

목차 (Table of Contents)

• 목차 ·······························································································1
• 그림목차 ························································································4
• 표 목차 ·························································································6
• 영문 초록 ······················································································7
• I. 막단백질의 발현, 정제과정 및 구조 분석·········································11
• A. 서론···························································································11
• (1) 막단백질 (Membrane protein)··················································11
• (2) 신데칸 (Syndecan)····································································13
• (3) 멜라노콜틴 수용체 (Human melanocortin receptor)··················15
• B. 본론 ··························································································19
• (1) Uniformly 15N labeled Syd4-eTC의 실험 방법 및 결과··············19
• a) Syd4-eTC의 발현········································································19
• b) Syd4-eTC의 분리 및 정제····························································22
• c) Syd4-eTC의 구조 분석································································36
• ① 용액상 핵 자기 공명 분광법을 이용한 구조 분석····························36
• ② 고체상 핵 자기 공명 분광법을 이용한 구조 분석····························37
• (2) Uniformly 15N labeled wt-hMC4R-TM2의 실험 방법 및 결과···42
• a) wt-hMC4R-TM2의 발현····························································42
• b) wt-hMC4R-TM2의 분리 및 정제················································44
• c) wt-hMC4R-TM2의 구조 분석····················································55
• C. 결론 및 토의·············································································59
• D. 시약 및 기구·············································································61
• E. 참고문헌··················································································68
• Ⅱ. NMR을 이용한 화학사고 피해 식물의 대사체 분석······················73
• A. 서론························································································73
• (1) 화학사고················································································73
• (2) 화학사고로 인한 식물피해 평가방법의 필요성····························79
• B. 본론························································································80
• (1) 화학사고 식물 피해 실험방법···················································80
• a) 시료의 채취·············································································80
• b) 화학물질에 노출된 갈참나무 잎·················································80
• ① 갈참나무 잎의 시료 전처리························································80
• ② Solution-State NMR 기법을 이용한 화학사고 식물피해 실험······82
• ③ HR-MAS NMR 기법을 이용한 화학사고 식물피해 실험···············84
• ④ Solid-State NMR 기법을 이용한 화학사고 식물피해 실험···········84
• c) 화학물질에 오염된 토양에서 자란 상추······································86
• ① 상추의 시료 전처리··································································86
• ② Solution-State NMR 기법을 이용한 화학사고 식물피해 실험······86
• ③ Solid-State NMR 기법을 이용한 화학사고 식물피해 실험···········88
• C. 결론 및 토의···········································································90
• D. 시약 및 기구···········································································93
• E. 참고문헌················································································96
• Ⅲ. 학술대회 및 논문 발표 실적····················································100