利用CD 和NMR 研究合成的神經胜肽片段 hNPY(18-26)之摺疊結構

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题名:	利用CD 和NMR 研究合成的神經胜肽片段 hNPY(18-26)之摺疊結構
其它题名:	Folding structure of synthesized neuropeptide segment hNPY [18-26] by CD and NMR
作者:	方智民;Fang, Chih-ming
贡献者:	淡江大學化學學系碩士班李長欣;Lee, Chang-shin
关键词:	神經胜肽;胜肽合成法;核磁共振;化學位移指數;擴散係數;NPY;SPPS;CD;NMR;CSI;diffusion coefficient
日期:	2009
上传时间:	2010-01-11 02:50:28 (UTC+8)
摘要:	神經胜肽是神經胜肽家族的成員之ㄧ，在1982 年，首次從豬的大腦出分離出來。神經胜肽家族的成員均含有36 個胺基酸，C 端皆修飾為NH2，並且含有大量的Tyr、Pro 和Arg。神經胜肽主要分布在中樞神經以及末梢神經系統中，並且控管許多重要的生理功能。本論文利用固相胜肽合成儀合成hNPY (18-26)，經由高效能液相層析儀純化，質譜儀卻認分子量，再利用圓二色旋光光譜和核磁共振光譜觀察不同比例TFE溶液下，二級結構的變化。此外，還利用DOSY 實驗觀察hNPY (18-26)在TFE 溶液中，自身結合的狀態。從CD 實驗中，發現hNPY (18-26)在70% TFE 溶液中最具有結構性；NMR 實驗判定hNPY (18-26)所有氫質子的化學位移。經由 NOE 限制條件分別計算hNPY (18-26)在10% D2O 與70% TFE-d3 溶液中的三維結構，可發現在10% D2O 溶液呈現無序纏捲，70% TFE-d3 溶液則在Arg19 到Ser22 出現螺旋結構；DOSY 實驗確定了hNPY (18-26) 在70% TFE-d3 溶液中主要以單體的型式存在。配合CD、NMR 以及結構計算，討論不同比例TFE 溶液下，hNPY (18-26)的結構差異，並且與完整的NPY 結構比較。 Neuropeptide Y (NPY), a member of the neuropeptide Y family of hormones, was first isolated from porcine brain in 1982 (Tatemoto et al. 1982). The peptides of this family all contain 36 amino acid residues, are C-terminally amidated, and contain a rather high content of tyrosine, proline and arginine residues. NPY is a very abundant neurotransmitter in the central and peripheral nervous system, and has been implicated in several functions. Herein, we synthesize neuropeptide segment hNPY (18-26) by SPPS, purified by RP-HPLC and made sure the molecular weight by Mass. The conformation and dynamics of hNPY (18-26) in difference solvent condition is studied by CD and NMR. We also defined the self-association state of hNPY (18-26) in TFE solution by DOSY experiment. The CD experiment, we can find the best helical conformation of hNPY18-26 at 70% TFE concentration. NMR experiments of COSY, TOCSY, ROESY, NOESY and HSQC were acquired. With NOE restrained structural calculation, the major structure of hNPY (18-26) in 10% D2O is random coil and form regular -helical structure between Arg19 and Ser22 in 70% TFE-d3. Combination od CD, NMR and XPLOR molecular calculation, we can investigate the conformational difference between 10 % D2O and 70% TFE-d3 and compare with native NPY.
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