立體電子效應，Proline 環構形效應及苯環與 Proline 作用力對 Polyproline 構形的影響

Study of polyproline conformation: stereoelectronic effects, ring puckering effects, and aromatic-proline interactions

林育如(Yu-Ju Lin)

指導教授 ： 洪嘉呈

國立清華大學/理學院/化學系/博士(2015年)

https://doi.org/10.6843/NTHU.2015.00576

摘要

脯胺酸 (Proline) 存在著環構形 (endo/exo) 及胜肽鍵異構化 (cis/trans) 的平衡。脯胺酸胜肽鍵的順反異構作用在蛋白質的折疊及對特定異構物的生化辨識過程中扮演很重要的角色，由於聚脯胺酸可以形成具有順式胜肽鍵的第一型 (PPI) 及具有反式胜肽鍵的第二型 (PPII) 螺旋結構，所以在研究脯胺酸胜肽鍵的順反異構上，聚脯胺酸是一個有價值的研究模型。最近研究顯示立體電子效應可以影響 PPII → PPI 的轉換速率，且以氟脯胺酸 (fluoroproline) 取代在聚脯胺酸上影響最大。在第二章中，我們將利用4號位子取代的氟脯胺酸置入在聚脯胺酸的氮端及碳端來探討立體電子效應對脯胺酸胜肽鍵順反異構化的影響，並且去探討末端的立體電子效應對聚脯胺酸構形的影響。利用圓二色光譜儀 (CD) 進行動力學研究，結果顯示碳端的立體電子效應對 PPII 轉換成 PPI 有較大的影響，嵌入三個 (2S,4R)-4- fluoroproline (Flp) 在碳端增加了 PPII 轉換成 PPI的活化能 (1.53 kJ mol-1)，而置入三個 (2S,4S)-4-fluoroproline (flp) 則降低了活化能 (4.61 kJ mol-1)。相反的，取代在氮端時， PPII 轉換成 PPI的活化能的差異僅有 -0.03 到 0.10 kJ mol-1。我們的結果證實立體電子效應對 PPII → PPI 轉換速率之影響是具有方向性的，亦符合了 PPII 摺疊的機構。
脯胺酸的環構形可藉由其環上不同的取代基來調控，由於立體電子效應，拉電子基取代在 4S 位子的脯胺酸衍生物傾向形成 C-endo 環構形及順式的胜肽鍵，及 PPI 螺旋結構。 4-Thiaproline (Thp) 傾向形成 endo 環構形及順式胜肽鍵，在第三章中，我們將利用一系列 Thp 取代的胜肽去探討 Thp 對聚脯胺酸的影響。我們合成了 P5ThpP5、P11、Thp7及 P7，並使用 CD 去鑑定結構，其結果顯示 Thp 取代不僅使 PPII 結構變不穩定，也降低形成 PPI 螺旋的傾向。我們也利用密度泛函理論 (DFT) 分析 Thp 與具有 Thp 的聚脯胺酸胜肽，其結果顯示 Thp endo/exo 構形的能量差非常小，此結果可能導致具有 Thp 的胜肽在溶液中會同時表現出 PPI 及 PPII 的結構。我們的實驗結果證實即使 Thp 的結構與脯胺酸相似，Thp 對聚脯胺酸的結構仍造成重大的影響。
在胜肽及蛋白質序列中，由於鄰近的芳香環對脯胺酸造成的作用力 (aromatic-proline interactions 或 proline-aromatic interactions) 使脯胺酸易形成順式的胜肽鍵。此作用力與芳香環的電子密度有密切的關係，富有電子的芳香環可形成較強的 aromatic-proline interaction 進而促進形成順式的胜肽鍵。在第四章中，我們嵌入天然的芳香族胺基酸 (F、Y、W) 於聚脯胺酸的碳端及氮端，去研究 aromatic-proline 效應對聚脯胺酸結構及 PPI ⟷ PPII 轉換動力學的影響。 CD 實驗結果顯示氮端的 aromatic-proline interaction對聚脯胺酸的結構影響比碳端的 aromatic-proline interaction 明顯。 PPI 的穩定度與 aromatic-proline interaction 的強度有關，且穩定度的排序以取代基代表則為 Y > W > F，然而 PPII 的穩定度顯示出相反的排序 F > W > Y 。隨時間變化的 CD 光譜所得動力學實驗結果顯示出芳香族胺基酸的置入效應對 PPII → PPI 的轉換是具有方向性的，相反的，對 PPI → PPII 的轉換是不具方向性的，其中芳香族胺基酸的疏水性側鏈扮演著一個重要的角色去影響 PPI 轉換成 PPII 的過程。我們的實驗結果顯示 aromatic-proline interaction 可用來調控 PPI 及 PPII 的穩定度；此外，我們亦提出 aromatic-proline interaction 可能發生在 PPI 折疊過程的後段之假設，因為其雖然增強了 PPI 結構之成分與緊密度，但無法加速折疊的過程。

關鍵字

立體電子效應 ； 脯胺酸 ； 聚脯胺酸 ； 環構型效應

並列摘要

Proline exists equilibria between endo/exo ring puckers and cis/trans peptide bond isomers. Prolyl cis/trans isomerization plays a critical role in protein folding and isomer-specific biochemical recognition. Since polyproline can form all-cis type I helices (PPI) or all-trans type II helices (PPII), it has been a valuable model to study the prolyl isomerization. Recent studies have shown that stereoelectronic effects influence the rate of PPII → PPI conversion and the fluoroproline substituted peptides have the most pronounced changes. In Chapter 2, we synthesized a series of host-guest peptides with 4-substituted fluoroproline incorporated into the N-terminus or the C-terminus and used a kinetic approach to explore terminal stereoelectronic effects on polyproline conformation. Time-dependent CD measurements revealed that incorporation of fluoroproline at the C-terminal end of polyproline has a large effect on PPII → PPI conversion, where a tri((2S,4R)-4-fluoroproline) sequence, (Flp)3, increases the transition barrier of PPII → PPI conversion by 1.53 kJ mol-1 while a tri((2S,4S)-4-fluoroproline) sequence, (flp)3, decreases the transition barrier by 4.61 kJ mol-1. In contrast, the same substitutions at the N-terminus only affect the transition barrier of PPII → PPI conversion by -0.03 to 0.10 kJ mol-1. Our results demonstrate stereoelectronic effects on PPII → PPI conversion are directional and provide an approach to establish the PPII → PPI transition mechanism.
The conformational equilibria between ring puckers can be modulated by substitutions on the proline ring. Implanting an electron-withdrawing group on the 4S position of proline prefers a C-endo pucker and a cis peptide bond due to stereoelectronic effects, and favors polyproline I (PPI) helices rather than polyproline II (PPII) helices by preorganization. 4-Thiaproline (Thp) favors an endo ring pucker and a cis peptide bond, and we incorporated Thp into polyproline peptides to explore its effects on the peptide conformation in Chapter 3. We synthesized a series of peptides, including P5ThpP5, P11, Thp7, and P7, and characterized their structures by CD spectroscopy. The results show that Thp substitutions not only destabilize PPII helices but also decrease the tendency to form PPI helices. The density functional theory (DFT) analysis on Thp and Thp-containing oligopeptide reveals that the energy difference between exo and endo ring puckers is small for Thp, i.e. Thp only slightly favors the endo pucker. Such a small energy difference could lead to the coexistence of PPI and PPII in solution for Thp-containing peptides. Our data demonstrate that although Thp possesses a structure similar to proline, it has a significant impact on polyproline conformation.
In a peptide or protein, the sequence with aromatic residues adjacent to proline residues shows a higher propensity in forming cis prolyl bonds due to aromatic-proline interactions or proline-aromatic interactions. The interactions are related to the electronegativity of aromatic- ring: an electron-rich aromatic ring relatively favored a cis amide bond. In Chapter 4, we incorporated aromatic amino acids (F, Y, W) into the N-terminal or the C-terminal end of polyproline to investigate aromatic-proline interactions on polyproline conformation and PPI ⟷ PPII interconversion kinetics. CD measurements reveal that the N-terminal aromatic-proline interaction significantly affects polyproline conformation more than the C-terminal aromatic-proline interaction. PPI stability is correlated with the strength of aromatic-proline interactions in the order of Y > W > F, while PPII stability is in an opposite order of F > W > Y. Time-dependent CD measurements reveal that aromatic-substitution effects are directional on PPII → PPI conversion but nondirectional on PPI → PPII conversion and the hydrophobicity of aromatic side chain may play a critical role in the conversion of PPI to PPII. Our data demonstrate that aromatic-proline interactions can modulate the stabilities of PPI and PPII conformations. Moreover, we proposed that aromatic-proline interactions may occur in the late stage of PPI folding process since the aromatic-proline interactions cannot speed the folding process.

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