對角帶電荷胺基酸側鏈長度對β-Hairpin穩定度影響

Abstract

β-Sheet是蛋白質中一種常見的二級結構，而股與股之間的作用力，例如：主鏈間的氫鍵或側鏈間的靜電作用力，都是可以使β-sheet結構穩定的主要作用力，然而，β-hairpin是反向平行β-sheet中最簡單的單元結構，因此為了探討帶電荷胺基酸側鏈長度對於β-sheet穩定度的關係，β-hairpin被設計作為實驗的模板。在β-hairpin中，股與股間位於正對或對角的胺基酸側鏈間的作用力都可以使得β-hairpin結構變得穩定，而對角位的胺基酸由於右手螺旋性質，使得在空間距離上變得接近，進而導致作用力的產生。在此研究中，我們探討了六種不同離子對的組合：Asp-Lys、Asp-Orn、Glu-Lys、Glu-Orn、Aad-Lys以及Aad-Orn，所有的胜肽都是利用固相合成法所合成，且利用高效能液化層析儀所純化，而2D-NMR的技術，包含了TOCSY、DQF-COSY以及ROESY則是被用來鑑定純化後的胜肽結構，β-hairpin的摺疊程度以及摺疊的自由能變化是由α質子的化學位移來判定。然而，實驗結果顯示出摺疊程度由大至小排列，依序為: HPDGluLys > HPDAadLys ≥ HPDGluOrn > HPDAadOrn > HPDAspOrn > HPDAspLys，此趨勢代表著離子對胺基酸側鏈愈長會使得β-hairpin結構的摺疊愈好、穩定度也愈高。

β-sheet is one of the common secondary structures in proteins. The β-sheet is mainly stabilized by backbone hydrogen bonds between adjacent strands and side chain interactions between amino acids on adjacent strands. To investigate how charged amino acid side chain length affects stability in β-sheets, a β-hairpin was studied as a model system, because β-hairpins are the simplest motif in antiparallel β-sheets. In a β-hairpin, lateral and diagonal cross-strand side chain-side chain interactions can stabilize the structure. The diagonal sites are spatially closer due to the right-handed twist. In this study, six diagonal cross-strand ion pairs were investigated: Asp-Lys, Asp-Orn, Glu-Lys, Glu-Orn, Aad-Lys, and Aad-Orn. All peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry and were purified by HPLC to at least 95% purity. After purification, the structures of the peptides were analyzed by 2D-NMR spectroscopy, acquiring TOCSY, DQF-COSY, and ROESY spectra. The β-hairpin population and folding free energy were determined from the chemical shifts of the α-protons. The results showed the fraction folded followed the trend HPDGluLys > HPDAadLys ≥ HPDGluOrn > HPDAadOrn > HPDAspOrn > HPDAspLys. This trend revealed that combinations with longer side chains gave higher hairpin stability.