精胺酸側鏈長度對β-Hairpin結構穩定度的影響及POG三肽對膠原蛋白穩定度與特異性的影響

Abstract

電荷胺基酸提供蛋白質靜電作用力(electrostatics)，而靜電作用力對於蛋白質結構的穩定性有重要的影響。β摺板(β-sheet)是蛋白質二級結構的其中一種並與許多蛋白質錯誤折疊的疾病相關。不同胺基酸對於形成β摺板有不同程度的傾向(propensity)，因此研究帶電荷胺基酸形成β摺板的傾向很重要。本研究著重在精胺酸(Arg)及其側鏈更長和更短的精胺酸衍生物(Agh, Agb, and Agp) 對於形成β摺板的傾向，以及序列位置的改變對其傾向的影響。β髮夾(β-hairpin)是β摺板最基本的構造。我們利用Fmoc主鏈保護的固相胜肽合成技術來合成不同序列位置帶有精胺酸及其衍生物的胜肽。 膠原蛋白三重螺旋(collagen triple helix)是由三條polyproline II螺旋(polyproline II helices)所組成。此研究中，我們將不同數目的POG三肽(POG triplets)插入三條具有異三聚體(heterotrimeric)性質且會形成polyproline II螺旋的胜肽序列中間。我們利用圓二色光譜儀做變溫實驗(thermal denaturation experiment)得出膠原蛋白三重螺旋的熔點(melting temperature)。此研究中，我們將不同數目的POG三肽(POG triplets)插入三條具有異三聚體(heterotrimeric)性質且會形成polyproline II螺旋的胜肽序列中間。我們利用圓二色光譜儀做變溫實驗(thermal denaturation experiment)得出膠原蛋白三重螺旋的熔點(melting temperature)。在變溫實驗中，熱平衡時間相當久，甚至接近熔點時的平衡時間需要2000分鐘之久。我們假設膠原蛋白三重螺旋在三聚體(trimer)與單體(monomer)間做平衡，並以膠原蛋白三重螺旋的加熱曲線來做fitting，得出膠原蛋白三重螺旋的熔點。在此實驗中，我們發現POG三肽對於polyproline II螺旋構造具有穩定的功效，然而隨著POG插入的數目增加膠原蛋白三重螺旋的特異性(specificity)會變差。本研究探討POG三肽的數目多寡對於膠原蛋白三重螺旋的穩定性與特異性之間的影響。

Electrostatics is important for protein structure stability. Charged amino acids contribute to these electrostatic interactions. β-Sheet is one of the protein secondary structures and is involved in various protein mis-folding diseases. Different amino acids have different propensities for β-sheet formation. Therefore, it is important to study the propensity of charged amino acids for β-sheet formation. This research focuses on the propensity of arginine (Arg) and arginine analogs with longer and shorter side chains (Agh, Agb, and Agp), and the position dependence of the corresponding sheet propensity. β-Hairpins are the simplest β-sheet structure. Hairpin peptides with arginine and arginine analogs incorporated at different guest sites were synthesized by Fmoc-based solid phase peptide synthesis. Collagen triple helix consists of three polyproline II helices. In this project, different numbers of POG triplets were inserted in the middle of all three heterotrimeric collagen triple helix forming peptides. Thermal denaturation experiments were used to determine the melting temperatures of the collagen triple helices. Thermal equilibration was found to be extremely long at temperatures around the melting transition, and up to 2000 minutes were required for reversible denaturation and renaturation. The melting temperatures were derived by fitting with the thermal denaturation curve, assuming a two-state trimer to monomer equilibrium. The POG triplet was found to stabilize the polyproline II structure in a collagen triple helix. Specificity was gradually decreased with the addition of more POG triplets. This study provides insights into how to use POG triplets to tune the stability and specificity of a collagen triple helix.