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막 단백질인 멜라노콜틴-4-수용체의 발현 및 정제의 최적화와 NMR을 이용한 구조 분석 원문보기

  • 저자

    朴裕根

  • 학위수여기관

    韓國外國語大學校 大學院

  • 학위구분

    국내석사

  • 학과

    화학과

  • 지도교수

    김용애

  • 발행년도

    2014

  • 총페이지

    75 p

  • 키워드

    transmembrane protein. melanocortin4receptor. solid-state NMR solid-state NMR probe;

  • 언어

    kor

  • 원문 URL

    http://www.riss.kr/link?id=T13538831&outLink=K  

  • 초록

    Optimized Expression, Purification and NMR structural studies of transmembrane peptide, human Melanocortin-4-receptor Human melanocortin-4 receptor (hMC4R) has a critical role in part of energy homeostasis. Heterozygous mutations in second trans-membrane domain of hMC4R relate in genetic cause of severe human obesity. In order to study the structure and function of these human trans-membrane proteins, it is important to prepare reasonable amounts of proteins. However, the preparation of human MC4R second trans-membrane peptide is seriously difficult and time-consuming. Overexpression and purification of membrane proteins was reported to be difficult due to their innate insoluble and toxic properties. Among the many difficulties, the most important is the difficulty in obtaining sufficient quantities of purified protein. Recently, we succeed to produce large amounts of the second trans-membrane domain from the wild-type hMC4R (wt-TM2) and mutant hMC4R (m-TM2). But, the purified protein contains decent amounts of KSI fragment somehow. Therefore, we spend a lot of time to optimize the purification schemes like fast protein liquid chromatography, dialysis, and chemical cleavage. The Circular dichroism and MALDI-TOF MS spectroscopy were used to identify the initial secondary structures and purity. In here, we demonstrate the optimization procedures to express and purify wild type-hMC4R TM2 and mutant-hMC4R TM2 peptides and NMR structural studies in different detergents to get high-resolution spectra. We obtained high resolution solution NMR spectra of hMC4R TM2. We will obtain its solid state NMR spectra and describe the structural difference between wild-type hMC4R (wt-TM2) and mutant hMC4R (m-TM2) in membrane-like environments.


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