離胺酸側鏈長度對在β-hairpin中與對面的α-氨基己二酸的離子對作用力的影響

Abstract

β摺板是一種重要的蛋白質二級結構。β摺板中相鄰兩股對面位置上常有異性電荷胺基酸存在，顯示了異性電荷胺基酸殘基間的作用力應是非常重要的。自然界中的帶電荷胺基酸如天冬胺酸、穀胺酸、賴胺酸、精胺酸有不同數目的疏水性亞甲基連接在主鏈上。因此，為什麼自然選擇了特定的側鏈長度，帶電荷胺基酸的側鏈長短又是否會影響相鄰兩股間的離子對作用力都是有趣的問題。研究表明相鄰兩股間對面位置的離子對的胺基酸殘基互換會影響β-hairpin的穩定度。之前的研究中探討過β-hairpin中靠近N端的帶負電荷的胺基酸殘基與對面位置上靠近C端的帶正電荷的胺基酸殘基間的作用力。在這裡，我們探討了相鄰兩股間對面位置的離子對的胺基酸殘基互換的影響，即靠近C端的帶正電荷的胺基酸殘基與對面位置上靠近N端的帶負電荷的胺基酸殘基間的作用力。 實驗中的胜肽是由固相胜肽合成法合成，並由高效液相層析儀純化至純度高於95%，由MALDI-TOF確認胜肽的分子量。完成純化後，我們藉由2D-NMR的技術來鑑定胜肽的結構，包含TOCSY、DQF-COSY以及ROESY。由α質子的化學位移可判定β-hairpin的折疊程度以及折疊的自由能變化。實驗結果顯示出摺疊程度從低到高依序為：HPTDapAad < HPTDabAad ~ HPTLysAad < HPTOrnAad。這個趨勢是胺基酸本身形成β摺板的傾向與離子對間的作用力共同造成的結果。相較於我們之前的研究，實驗結果表明離子對在穩定β摺板上是有位相的偏好的。

β-Sheets are one of the important secondary structures in proteins. Oppositely charged residue pairs are frequently found at lateral positions of an antiparallel β-sheet, which suggests that interactions between such residues may be important for the stability of antiparallel β-sheets. The naturally existing charged amino acid residues Asp, Glu, Lys and Arg have different numbers of hydrophobic methylenes linking to the backbone. Why did nature choose these specific side chain lengths and whether the charged amino acids side chain length have an effect on cross strand ion pairing interactions are therefore interesting questions. Studies have shown that swapping the residues in a lateral ion pair changed the stability of proteins. Negatively charged residues incorporated closer to the N-terminus with lateral interactions with positively charged residues incorporated closer to the C-terminus have been previously studied in a β-hairpin by the Cheng group. Herein, we specifically studied the effect of swapping the position of cross strand lateral ion pairing residues, with the positively charged Lys analogs closer to the N-terminus with lateral interactions with the negatively charged residue (S)-3-aminoadipic acid (Aad) closer to the C-terminus. The peptides were synthesized by solid phase peptide synthesis using Fmoc-based chemistry and were purified by HPLC to higher than 95% purity. The identity of the peptides were confirmed by MALDI-TOF. The peptides were analyzed by 2D-NMR spectroscopy, including TOCSY, DQF-COSY, and ROESY spectra. The β-hairpin structure of the peptide was confirmed by chemical shift deviation, 3JHNα, and characteristics NOE connectivities. The β-hairpin population and folding free energy were determined from the chemical shift deviations of the α-protons. The fraction folded population follow the trend HPTDapAad < HPTDabAad ~ HPTLysAad < HPTOrnAad. The trend was the result of the β-sheet propensity of the constituting residues and the ion pairing interaction energy. Compared to our previous study, the results showed that ion pairs have an orientation preference for stabilizing an antiparallel β-sheet.