帶負電荷(i)與正電荷(i+3)的胺基酸鏈內離子對作用力對膠原蛋白穩定度的影響

Abstract

膠原蛋白支撐身體結構。 序列主要由Gly-Xaa-Yaa重複單位組成。 帶電荷胺基酸比預期中還要更頻繁地出現於膠原蛋白序列之中。 顯示帶電荷胺基酸對膠原蛋白的重要性。 膠原蛋白三股螺旋的設計是基於ABC形式的異元三聚體， 藉此可避免其他不必要的作用力干擾。 利用圓二色光譜儀來測量膠原蛋白三股螺旋的熱變性與熱重組過程， 並從中得到的熱變性數據推導出熱力學參數， 再利用雙突變循環分析方法探討同一條鏈上的離子的作用力。 實驗結果顯示在Y位置的Glu-Lys和Glu-Arg離子對有作用力。 Glu-Lys和Glu-Arg在Y位置上有比在X位置上更多的作用力。 這些結果告訴我們離子對對膠原蛋白三股螺旋的影響。 平行β-sheet 是蛋白質主要的二級結構並常見於阿茲海默症患者腦內。帶相反電荷的胺基酸常出現在平行β-sheet中， 顯示帶電荷胺基酸對平行β-sheet的重要性。不同於非平行β-sheet， 平行beta-sheet需要合成C/N端linkers以連結住兩段beta-strands。在成功合成C端和N端linkers 後即可開始beta-strands的合成。 平行β-sheet合成後是以2D NMR並取得TOCSY, DQF-COSY,和 ROESY圖譜。

Collagen is the substance that holds the whole body together. Collagen consists of the repeating Xaa-Yaa-glycine sequence. Collagen contains more charged residues than expected. Therefore, investigating the effect of the charged residues on collagen triple helix stability should unravel the origin of the stability of collagen. The collagen triple helices were designed based on an ABC-type heterotrimer collagen. All peptides were synthesized by Fmoc-based solid phase peptide synthesis. After purification, thermal denaturation was monitored by circular dichroism spectroscopy. The thermal denaturation data was used to derive the melting temperature and unfolding free energy of the collagen triple helices. Double mutant cycle was used to determine the intrachain interaction energies. Stabilizing interaction in the collagen triple helix was observed for pairs Glu-Lys between two adjacent Y positions. Destabilization was apparent for pairs Glu-Lys, Glu-Arg, and Asp-Lys between two adjacent X positions. In general, incorporating ion pairs Glu-Lys at the adjacent Y positions was more stabilizing compared to same ion pairs at the adjacent X positions. The results offered insights for the effect of intrachain ion pairing interactions on collagen triple helix stability. β-Sheet is one of the major secondary structures in proteins. All peptides were synthesized using Fmoc-base solid phase peptide synthesis. The linkers were synthesized using solution phase organic synthesis. The experimental pseudopeptide contained two parallel acetyl capped strands connected by a diamine linker at the C-termini. The fully folded reference pseudopeptide contained two parallel strands connected by a diamine linker and diacid linker at the C-termini and the N-termini, respectively. The fully unfolded reference peptide consists of the acetyl capped single strands without any linker on either end. The pseudopeptides and peptides were analyzed using 2D NMR (TOCSY, COSY, and ROESY). The experimental pseudopeptides P1QLysGlu, P1QLysAla, and P1QAlaAla were synthesized, purified, and 2D NMR spectra were acquired. The fully folded reference pseudopeptides P1QFLysGlu, P1QFLysAla, P1QFAlaGlu, and P1QFAlaAla were synthesized, purified, and 2D NMR spectra were acquired. Fully unfolded reference peptides P1Q1Lys and P1Q2Glu were synthesized, purified, and 2D NMR spectra were acquired.