利用CD及1H NMR研究hNPY類似物的摺疊結構

Folding structure of analogues of hNPY by CD and 1H NMR

黃品鈞(Pin-Chun Huang)

指導教授 ： 李長欣

淡江大學/理學院/化學學系碩士班/碩士(2008年)

https://doi.org/10.6846/TKU.2008.01111

摘要

人類神經胜肽Y(Human Neuropeptide Y, hNPY)在水溶液中具有α-螺旋結構，且奈米莫爾濃度(nM)下結構為單體，毫莫爾濃度(mM)時則為雙體。其結構有特定作用機制：單體先與膜微脂粒(membrane micelle)結合後進一步與GPCRs(G protein-coupled receptors)作用。固相胜肽合成法(Solid Phase Peptide Synthesis)合成hNPY片段序列hNPY【11-36】，經由高效能液相層析儀純化。再由合成hNPY【18-36】，此兩胜肽經由質譜儀確認分子量，再用圓二色光譜(Circular Dichroism)觀察不同比例TFE溶液下，二級結構的變化，選擇100%水溶液與50%TFE兩溶液做結構測量與討論。用二維核磁共振(two-dimensional NMR)的光譜: TOCSY、NOESY、[1H, 13C]-HSQC及光譜判定(Assignment)和光譜循序判定(Sequential assignment)，各別判定兩溶液之殘基質子與13碳的化學位移。經由NOE限制條件分別計算兩溶液之3D結構，100%水溶液呈現鬆散不規則結構，50%TFE溶液283K及310K分別在24Leu~31Ile及25Arg~31Ile出現α-螺旋結構。由DOSY光譜可得知hNPY【11-36】水溶液結構介於雙體及三體間，hNPY【18-36】 50%TFE溶液結構介於單體及雙體間。
配合CD、CSI(Chemical Shift Index)、NMR、DOSY、結構計算，討論50%TFE溶液下兩種溫度之結構差異，並與NPY結構及文獻比較。

關鍵字

核磁共振 ； 圓二色光譜 ； 固相胜肽合成 ； NPY

並列摘要

Human Neuropeptide Y(hNPY) has a well-defined α-helical structure in solution, and is monomer at nM concentration, dimer at mM concentration. It has specific binding mechanism: First, the monomer structure binds with membrane micelle, and further interacts with G protein-coulpled receptors(GPCRs).
We synthesize neuropeptide fragment hNPY【11-36】by solid phase peptide synthesis, purified by RP-HPLC. Synthesize neuropeptide fragment hNPY【18-36】from and made sure the molecular weight by Mass. The conformation and dynamics of hNPY【18-36】in difference solvent condition is studied by CD and 2D NMR experiment.
2D NMR experiments of TOCSY, NOESY, and [1H, 13C]-HSQC were acquired. With NOE restrained structural calculation, the major structure of hNPY【18-36】in 100% H2O is random coil and form regular α-helical structure between 24Leu and 31Ile, 25Arg and 31Ile in 50%TFE/50% H2O 283K and 310K separately.hNPY【11-36】structure in 100%H2O between dimer and trimer by DOSY spectrum,
hNPY【18-36】structure in 50%TFE/50% H2O between monomer and dimer.
Combination of CD, NMR, and XPLOR molecular calculation, we can investigate the conformational difference between 100%H2O and 50%TFE /50%H2O, and compare with native NPY and paper.

並列關鍵字

NMR ； CD ； NPY ； SPPS

參考文獻

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<3> McLean, L. R., Buck, S. H., Krstenansky, J. L. “Examination of the Role of the Amphiphatic α-Helix in the Interaction of Neuropeptide Y and Active Cylic Analogues with Cell Membrane Receptors and dimyristolphosphatidylcholine” Biochemisry, 1990, 29, 2016-2022.