雞絨毛蛋白片段HP36是一個具有36個胺基酸的小型螺旋蛋白，主要構形由三個α-螺旋所組成，由於具有體積小、結構穩定、摺疊快速等特性，使得HP36成為蛋白質摺疊的熱門研究模型。在先前的研究結果中顯示HP36在未摺疊態 (unfolded state) 下仍然具有部分的二級結構存在，這項特點被認為是HP36能夠快速進行摺疊的關鍵。在本研究中，我們設計五個不同位置的組合 (44-48、44-49、56-59、57-60、68-72號位) 分別在HP36的三個α-螺旋上置換半胱胺酸 (cysteine)，並利用間二甲苯基團 (m-xylene) 進行交聯 (cross-link) 將α-螺旋結構固定。由Far-UV CD光譜、NMR光譜及螢光光譜顯示所有胜肽都具有與原生態 (wild-type) 相似的構形；利用變性實驗測量穩定度與連動性 (cooperativity) 後，其結果指出交聯能夠使胜肽穩定度上升，置換半胱胺酸則會使胜肽穩定度下降，而藉由連動性的變化我們可以推測在未摺疊態中殘存的二級結構可能位於α1螺旋的碳端 (C-terminus) 以及α3螺旋上，α2螺旋則對結構展開 (unfold) 過程十分重要。由Discovery Studio程式在CHARMm力場下進行分子動力學 (molecular dynamics) 的模擬結果同樣顯示所有胜肽都具有與原生態相似的構形，而殘基間作用力的計算結果證明半胱胺酸的強烈疏水性是造成胜肽穩定度下降的主因。特別的是，α3螺旋 (68-72) 在進行交聯後能使胜肽整體穩定度顯著上升，此結果值得進一步研究是否與先前研究中提到的α3-α2螺旋之間的連動穩定效應有關。

The helical subdomain of villin headpiece, HP36, is one of the smallest naturally occurring cooperatively folded proteins. Because of its small size, rapid folding rate and simple three-helix topology, HP36 has been a popular model for theoretical and computational study of protein folding. Many studies have suggested that there are residual secondary structures of HP36 in the unfolded state, and considered it to be the key of fast-folding. In our study, five different combinations of double cysteine mutation were carried out on the three helices of HP36, and m-xylene was used as a cross-linker to staple the α-helix structure. Far-UV CD, NMR, and fluorescence spectra indicate that all of the mutants fold into a similar structure to that of the wild-type. By denaturation experiments, stability and cooperativity of the proteins are determined. The results show that cross-linking stabilizes the proteins, while cysteine mutation destabilizes the proteins. From the decreases of cooperativity, we conclude that α3 helix and the C-terminus of α1 helix likely maintain residual secondary structures in the unfolded state, and α2 helix may play an important role in HP36 unfolding. The final structures from MD simulation under CHARMm force field are similar to that of wild-type, and the calculated interaction energy between the residues reveals that the hydrophobicity of cysteine results in the destabilization of the proteins. Remarkably, the mutant with cross-linked α3 helix demonstrates the high stability, and the result may be related to the α3-α2 cooperative stabilizing which was reported in the literature.

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