- 學位論文
(I) 立體電子效應對雞絨毛蛋白 (HP36) 結構影響之探討; (II) 電子自旋標記聚脯胺酸的合成及電子自旋共振光譜之量測
(I) Stereoelectronic Effects on Villin Headpiece Subdomain (HP36) Structure and Stability; (II) Synthesis and EPR Measurements of Spin-labeled Polyproline Peptides
摘要
第一部分為研究立體電子效應在雞絨毛蛋白上的作用。脯胺酸 (proline) 會形成 exo 或是 endo 的構形，當4R (反式) 位置有拉電子基取代時偏好 Cγ exo 構形，而 4S (順式) 則偏好 Cγ endo 構形，造成這樣的原因我們稱之立體電子效應。在小蛋白 Trp-cage 的研究例子中，增加脯胺酸的 exo 構形傾向時，其螺旋結構穩定度會增加，而此脯胺酸是位於 Trp-cage 螺旋的碳端。為了探討當立體電子效應發生於螺旋的氮端時，是否也能穩定螺旋結構，因此我們利用具有36個胺基酸的雞絨毛蛋白 (HP36) 來作為研究之蛋白模型。經由 CD 量測之變溫實驗與化學誘導變性實驗，我們發現當脯胺酸置換成 4R 位置有拉電子基之脯胺酸衍生物 (4R-proline derivatives) 時， HP36 的穩定度會略為下降，這是因為在 HP36 中的 Pro62 會與 Trp64 有著 proline-aromatic 作用力，所以這樣的置換會破壞 proline-aromatic 作用力，並且也會增加其立體空間障礙，導致HP36 的不穩定。 第二部分為研究聚脯胺酸第二型結構 (polyproline type II, PPII) 的EPR 光譜規則建立。在α-螺旋胜的例子中，已成功利用兩個電子自旋標記接在胜上，並利用 EPR 光譜量測胺基酸間之距離。在此我們合成一系列的聚脯胺酸胜，並成功地製備了含有電子自旋標記的胜，且進一步用 EPR 光譜量測兩個電子自旋標記之間的距離；在經過一系列的數學運算後，分析計算 EPR 光譜以得到電子自旋標記間之距離。利用這樣的方法計算出 PPII 中，每個胺基酸的距離為 0.31 nm ，這與 PPII 模型的理論值相同，所以我們成功的利用 EPR 光譜來量測 PPII 結構中胺基酸之間的距離。
並列摘要
The first part of this thesis is about stereoelectroic effects on villin headpiece subdomain (HP36). Proline can form either a Cγ exo ring pucker or a Cγ endo ring pucker. An electron-withdrawing substituent at the 4R position of proline makes proline favor an exo ring pucker, while that at the 4S position favors an endo ring pucker due to stereoelectronic effects. Stereoelectronic effects have been shown to tune the stability of a small helical protein, the Trp-cage. In the Trp-cage, increasing the exo ring pucker propensity at C-terminus of the helix can stabilize the protein. To investigate stereoelectronic effects on the N-terminus of an α-helix, we use a 36-residue helical protein, the villin headpiece subdomain (HP36), as our protein model. A few proline derivatives including (4R,2S)-4-hydroxyproline, (4R,2S)-4-methoxyproline, and (4R,2S)-4-fluoroproline were used to replace the proline in HP36. Thermal unfolding and urea denaturation measurements by CD spectroscopy have shown that the mutation destabilized the protein. Our primary results suggest that there are not only stereoelectronic effects but also proline-aromatic interactions and steric effects affecting the structure in this region. The second part of this thesis is about establishing the PPII spectrum rule by EPR. We utilized double spin labels to get the dipole-dipole moment which can be used to calculate the distance between two spin labels. A series of polyproline peptides were designed and synthesized for this purpose. The spin-labeled peptides were successfully prepared for EPR measurements. An mathematical method was applied to calculate the distance between amino acids in PPII helices. The value of 0.31 nm between two adjacent residues is consistent with the theoretical distance for a PPII helix.
參考文獻
延伸閱讀
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