聚脯胺酸胜肽的探討：末端帶電荷胺基酸對結構之影響、與人類Pin1 WW區間的結合親和力及作為酯類水解酶之設計

Study of polyproline peptides: the effects of terminal charged residues on conformation, the binding affinity for human Pin1 WW domain and the design of artificial esterases

黃魁彥(Kuei-Yen Huang)

指導教授 ： 洪嘉呈

國立清華大學/理學院/化學系所/博士(2019年)

摘要

聚脯胺酸螺旋有兩種主要的異構物，包含全為順式胜肽鍵的第一型 (PPI) 與全為反式胜肽鍵的第二型 (PPII) 聚脯胺酸螺旋結構，此二構形可幫助我們了解脯胺酸胜肽鍵異構的原因。PPI與PPII的穩定性可被許多效應所調控，但影響聚脯胺酸結構穩定性的原因仍未被完全了解，論文第二章中，將以不同帶電荷胺基酸置入聚脯胺酸結構的兩端以探討其對結構的影響。圓二色光譜儀 (CD) 的量測中發現正電荷胺基酸在碳端或負電荷胺基酸在氮端皆可以穩定PPII螺旋結構，而且其中以前者影響甚劇，代表著靜電作用力具方向性且正電荷胺基酸與碳端羧酸基的作用力穩定PPII結構之效果較負電荷胺基酸與氮端胺基的作用力來得大。相反地，在氮端正電荷胺基酸比負電荷胺基酸適合穩定PPI結構。在PPI結構最佳化模型計算結果中，發現正電荷胺基酸在碳端展現出與其他胜肽相反的偶極矩方向，可以說明極性反轉可能為不傾向形成PPI螺旋的原因。此部分結果顯示，末端胺基酸的靜電作用力可以大幅影響聚脯胺酸結構的穩定性，對日後設計聚脯胺酸構形為基底的生物材料的應用上有顯著的幫助。
　　此外，Pin1為細胞循環中重要的順反異構酶，若Pin1功能的過度或缺乏調控分別會造成癌症或阿茲海默症，故Pin1功能的調控為當今研究的重點之一。Pin1蛋白有兩個區間，其一為WW區間，負責辨認與結合含有磷酸化絲/蘇胺酸-脯胺酸位元(pS/pT-P motifs)，另一PPIase區間則負責催化此位元之胜肽鍵順反異構化進而管控細胞循環。論文第三章中，我們根據含磷酸化胺基酸之聚脯胺酸構形配體Myt1-T412 [PPA(pT)PP] ，將其pS/pT-p motif中的脯胺酸置換成帶有不同的拉電子基的4號碳取代脯胺酸衍生物而合成一系列短胜肽配體，想要藉此尋找Pin1的抑制劑。等溫滴定微量熱儀 (ITC) 與螢光異向性 (FA) 的分析中發現(2S,4R)-4-fluoroproline可以增加其與Pin1 WW區間的結合能力，CD光譜則發現與Pin1 WW區間結合能力越好的胜肽越傾向形成PPII螺旋結構，核磁共振光譜儀 (NMR) 的1H-15N HSQC圖譜與結構模擬也發現(2S,4R)-4-fluoroproline與Pin1 WW區間上的結合位點中的34號色胺酸有著較強的C−H···π作用力，這些結果提供了設計Pin1抑制劑的新資訊。
　　最後，由於天然酵素難以取得且昂貴，加上其不穩定性，研發符合經濟效益之人工酵素於近期如火如荼地展開。論文的第四章中，設計了連結含催化二聯體〈組胺酸-組胺酸或絲胺酸-組胺酸〉(His-His or His-Ser) 的聚脯胺酸骨架與自組裝蛋白之新人工水解酶 (H2H5和H2S5) 。CD光譜顯示，從pH 5到10的過程中，此二胜肽的二級結構由隨機螺旋轉為β-摺板構形。在pH 9.0的狀況下，以穿透式電子 (TEM) 及原子力學顯微鏡 (AFM) 皆可觀察到自組裝的奈米纖維網狀結構；傅立葉轉換衰減全反射红外光譜儀中也可以觀測到其β-摺板構形的低頻與高頻振動模式。此結果顯示，β-摺板構形與纖維結構的多寡也與催化能力有高度相關。利用p-nitrophenyl acetate (p-NPA) 作為酯類水解反應的受體，在pH 9.0的狀態下，我們發現H2H5具有高的酯類水解反應催化效率，而H2S5則是在pH 10.0的狀態下有高催化效率。此外，H2S5也對於p-nitrophenyl-(2-phenyl)-propanoate (p-NPP) 及 p-nitrophenyl-methoxy-acetate (p-NPMA) 有較高的親和力，此部分的結果提供了新的人工水解酶的設計方法。

關鍵字

脯胺酸 ； 聚脯胺酸構形 ； 結合親和力 ； 人工酯類水解酶

並列摘要

Polyproline is a useful system for better understanding proline isomerization because it exists predominantly as two forms, all-cis polyproline I (PPI) and all-trans polyproline II (PPII) helices. In Chapter 2, we study the impacts of terminal charged residues on polyproline conformation. Circular dichroism (CD) measurements show that a cationic residue at the C-terminus or an anionic residue at the N-terminus increases the stability of a PPII helix; in particular, the C-terminal cationic residues impose an enormous impact on PPII stability, suggesting that the stabilization effect of electrostatic interactions on PPII is directional. In contrast, incorporating a cationic residue seems more favorable than adding an anionic residue into the N-terminus because the cationic residue can stabilize PPI. The predicted dipole moments from optimized oligopeptide models reveal that the macrodipole of the peptides with a cationic residue at the C-terminus exhibits the opposite direction to that of other peptides in the PPI conformation, suggesting that such a dipole distortion may cause these peptides to disfavor PPI helices. This study provides useful information to the design of polyproline-based scaffolds for biomedical applications.
Over- and under-regulation of Pin1, which modulates the cis-trans isomerization of prolyl bonds in cell cycle, may lead to cancers and Alzheimer’s diseases, respectively. Thus, the regulation of Pin1 function has become an attractive topic. The WW domain of human Pin1 can recognize the phosphoserine/ phosphothreonine-proline (pS/pT-P) motifs, while its PPIase domain catalyzes the cis/trans isomerization of prolyl bonds to regulate the cell cycle. In Chapter 3, on the basis of the ligand Myt1-T412 [PPA(pT)PP], we synthesized several phosphopeptides in which the proline residue in the pT-P motif was replaced with various 4-substituted proline derivatives to develop a better inhibitor of Pin1. Isothermal titration calorimetry (ITC) and fluorescence anisotropy (FA) analyses show that the replacement of proline with (2S,4R)-4-fluoroproline increases the binding affinity of the peptide. CD measurements suggest that a more PPII-like structure of phosphopeptides makes them bind to the WW domain more tightly. Chemical shift perturbation experiments also indicate that (2S,4R)-4-fluoroproline interacts with Trp34 of the WW domain in the binding site. Results of molecular modeling further suggested that a strong C−H···π interaction induced by (2S,4R)-4-fluoroproline is important in enhancing the affinity of the peptide for the WW domain. The results provide new valuable information for designing and developing effective inhibitors of human Pin1.
Besides, researchers have been using different approaches to develop artificial enzymes with stability and availability due to the high cost and instability of natural enzymes. In Chapter 4, we combined a polyproline scaffold containing a catalytic dyad of His-His or Ser-His with a self-assembling peptide MAX1 to design new catalysts (H2H5 and H2S5) for ester hydrolysis. CD measurements indicate that these peptides have a conformational change from random coils to β-sheets upon increasing the pH values from 5 to 10. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) shows that these two peptides can self-assemble into similar network fibrils. The attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectra display specific vibration modes corresponding to the β-structures at pH 9.0. The self-assembled β-structures have a positive effect on the catalytic efficiency for ester hydrolysis. Using p-nitrophenyl acetate (p-NPA) as a substrate, H2H5 exhibits a relatively high catalytic efficiency at pH 9.0, while H2S5 has an even better activity at pH 10.0. Moreover, the H2S5 has a better efficiency on catalyzing p-nitrophenyl-(2-phenyl)-propanoate (p-NPP) and p-nitrophenyl-methoxy-acetate (p-NPMA) than H2H5 due to that it has a higher affinity for these substrates. The results demonstrate that combining the polyproline scaffold with the self-assembling peptide can achieve a high catalytic activity on ester hydrolysis, providing a new design for the development of effective artificial enzymes.

並列關鍵字

Proline ； Polyproline conformation ； Binding affinity ； Artificial esterase

參考文獻

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