| 摘要: | 人類神經胜肽Y(human neuropeptide Y；hNPY)在水溶液中具有alpha-螺旋結構，而在低濃度情況下為單體(monomer)結構，在高濃度時則為雙體(dimer)結構。其結構有特定作用機制：先形成單體再與膜微脂粒(membrane micelle)結合，再進一步與GPCRs(G protein-coupled receptors)作用。
我們利用圓二色光譜(Circular Dichroism)觀察 hNPY【15-29】在不同濃度的TFE水溶液中，其二級結構的變化。並選擇100％ H2O和50％ TFE / 50％ H2O兩種水溶液做結構的測量與討論。再利用二維核磁共振(2D NMR)實驗：COSY、TOCSY、NOESY、[13C, 1H]-HSQC等光譜，完成1H-NMR光譜的序列判定(assignment)之後，可以得到1H和13C的化學位移指標，並能預測 hNPY【15-29】之二級結構形成的區域。
藉由NOE之距離限制條件(NOE distance constraints)，利用XPLOR軟體進行結構運算以及分子動態模擬焠熄(molecular dynamics simulation annealing)及能量最小化和結構精煉化(refinement)的過程後，得到20個hNPY【15-29】的模擬構型之三度空間結構。
我們利用脈衝梯度磁場核磁共振(PFG-NMR)實驗，探討 hNPY【15-29】在溶液中是否有聚集(aggregation)的現象，亦即觀察其是否有雙體(dimer)或其他多聚物(oligomer)的形成。並利用氫氘交換實驗來觀測 hNPY【15-29】是否有分子內氫鍵的形成以穩定其分子結構。
我們綜合CD實驗、CSI(chemical shift index)、分子動力學模擬實驗所得到的結果，可以確定 hNPY【15-29】在100％ H2O中不完全是無序纏繞(random coil)的結構，可能還有其他規則性的二級結構存在；而在50％ TFE / 50％ H2O水溶液中，在殘基Y20到殘基L24的區域會形成alpha-螺旋結構，而且是以單體結構存在於TFE水溶液中。另外我們發現溫度的變化也會影響 hNPY【15-29】之螺旋結構的穩定性，亦即在低溫下的結構比較緊密；在高溫下的結構比較鬆散。
Human neuropeptide Y (hNPY) has a well-defined alpha-helical structure in solution, and is monomer at low concentration, but is dimer at high concentration. It has specific binding mechanism : First, the monomer structure binds with membrane micelle, and further interacts with G-protein coupled receptors (GPCRs).
We probe into the folding conformation of hNPY【15-29】in difference solvent condition, 100％ H2O and 50％ TFE / 50％ H2O by CD and 2D NMR experiment. Chemical shift index (CSI) of 1H and 13C were acquired after finishing assignments of 2D NMR spectroscopy of COSY, TOCSY, NOESY, and [13C, 1H]-HSQC. By using CSI, we can predict the secondary structure of hNPY【15-29】.
NOE distance constraints were determined from NOESY spestra. We used XPLOR for structure calculations, including molecular dynamics simulation annealing and energy-minimized process. Twenty refined hNPY【15-29】conformations were resulted from structure simulation.
We use PFG-NMR to estimate the dissociation and self-association of hNPY【15-29】in aqueous TFE. Also, we investigate whether or not hNPY【15-29】has intramolecular hydrogen bond by performing hydrogen /deuterium exchange experiment.
Combination of CD, 2D NMR, and structure calculations, PFG-NMR, we assured that hNPY【15-29】assumes not only a conformation of random coil but also other regular secondary structure in 100％ H2O; assumes a monomeric and alpha-helical structure in 50％ TFE / 50％ H2O. At lower temperature, the structure of hNPY【15-29】is more compact while at higher temperature, it is more flexible. |
