帶正電荷(i)與負電荷(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

膠原蛋白是動物體內含量最豐富的蛋白質，在組織發育與再生的過程中扮演重要的角色。膠原蛋白由三條左手螺旋的polyproline II螺旋互相纏繞而成，最後形成右手三股螺旋的結構。序列主要由Gly-Xaa-Yaa三聯重複單元組成。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，並且序列中X位置與Y位置也不是隨機分布的，顯示帶電荷胺基酸對於膠原蛋白的重要性。帶電荷胺基酸透過形成鏈間或鏈內的成對作用力來參與膠原蛋白三股螺旋的生成。同元三聚體的膠原蛋白三股螺旋中潛在的鏈間或鏈內成對作用力於先前已經被研究過。本篇主要研究膠原蛋白鏈內的離子對作用力對於穩定度的影響。在此，膠原蛋白三股螺旋的設計是基於ABC形式的異元三聚體，這樣一來可以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對之間是否存在作用力。實驗結果顯示，大部分帶相異電荷的離子對在膠原蛋白三股螺旋結構中存在著作用力，其中以在Y位置的Lys-Glu離子對的作用力最強。Lys-Asp離子對不論X位置或Y位置均沒有明顯的作用力。此外也 發現，同樣的離子對在Y位置會比在X位置具有更強的作用力。這些研究結果顯示，帶有吸引力的離子對作用力確實存在於膠原蛋白三股螺旋中，並且對於膠原蛋白三股螺旋結構的穩定性有所幫助。

Collagen is the most abundant protein in animals, providing a structural framework during tissue development and regeneration. Collagen forms a right-handed triple helix that contains three left-handed polyproline II-like helices. The common sequence of collagen triple helix consists of Gly-Xaa-Yaa triplets. Charged residues occur more frequently than expected and are non-randomly distributed between the Xaa and Yaa positions in collagen chain. Charged residues participate in collagen triple helix formation through interchain/intrachain pairwise interactions. Potential interchain/intrachain pairwise interactions were previously studied in homotrimers. This research focuses on the effect of intrachain ion pairing interactions between oppositely charged residues on collagen triple helix stability. The collagen triple helices were designed based on an ABC heterotrimer to avoid undesirable complicating interactions. All peptides were synthesized by Fmoc-based solid phase peptide synthesis. The thermal denaturation/renaturation of collagen triple helices was monitored by circular dichroism (CD) spectroscopy. The thermodynamic parameters Tm, ∆HTm, ∆STm, and ∆Gunfold were derived from the thermal denaturation data. Double mutant cycle was used to determine the potential intrachain ion pairing interaction. The results showed that almost all of the oppositely charged residue pairs exhibited a stabilizing ion pairing interaction. The most stabilizing interaction was observed for Lys-Glu between two adjacent Y positions. No interactions were seen for Lys-Asp between two adjacent X or Y positions. Additionally, the same charged pair seemed to provide more stabilizing interaction energy at Y positions compared to that at X positions. These results demonstrate that attractive ion pairing interactions within collagen chains contributes to the stabilization of collagen triple helix formation.