側鏈長度對β-Hairpin離子作用力及雙甲基化精胺酸對核糖核酸辨識與細胞穿透之影響

Abstract

離子作用力對於穩定蛋白質結構及核糖核酸辨識扮演相當重要的角色，主要由帶相反電荷的胺基酸所形成。自然界中帶電荷胺基酸具有不同的側鏈長度，例如負電荷胺基酸中，Asp及Glu側鏈分別具有一個與兩個疏水性亞甲基團，非自然界負電荷胺基酸Aad則具有三個亞甲基團。為研究負電荷胺基酸側鏈長度對於β-hairpin結構穩定中之離子作用力的影響，遂設計合成出一系列帶有不同負電荷胺基酸Zbb (Asp, Glu及Aad)之β-hairpin胜肽 (HPTZbbArg)。利用二維核磁共振光譜可確認胜肽HPTAspArg、HPTGluArg及HPTAadArg所形成之β-hairpin結構。比較所合成之胜肽與控制組胜肽中各殘基α氫原子化學位移差異可計算出胜肽分子之摺疊比例與自由能。β-Hairpin跨股側向離子作用力之能量大小以雙突變循環分析，結果顯示：Aad-Arg > Glu-Arg ≈ Asp-Arg，可知側鏈愈長的負電荷胺基酸，其跨股側向離子作用力之能量愈大。 人類免疫缺陷病毒Tat蛋白中47-57區域對於專一性辨識TAR RNA及穿透細胞非常重要。Tat蛋白與TAR RNA之結合對於人類免疫缺陷病毒基因組複製是不可或缺的。為研究Arg雙甲基化對於TAR RNA辨識及穿透細胞之影響，將Tat蛋白中47-57區域之六個Arg分別取代成ADMA(不對稱雙甲基化)及SDMA(對稱雙甲基化)，利用膠體電泳位移分析Tat衍生物對TAR RNA之結合能力與專一性，並利用流式細胞儀評估Tat衍生物穿透Jurkat細胞膜且進入細胞內部的效率。

Ion pairing interactions play important roles in protein stability and RNA recognition. Ion pairs are formed between a pair of oppositely charged amino acids. Interestingly, natural charged amino acids have different number of hydrophobic methylenes on their side chains. For negatively charged residues, Asp has one methylene and Glu has two methylenes. The analogous non-encoded negatively charged amino acid, Aad, contains three methylenes. To study the effect of negatively charged amino acid side chain length on cross strand ion pairs in β-sheets stability, a basic β-hairpin model HPTZbbArg was designed. Zbb denotes the negatively charged residues. The hairpin structure for peptides HPTAspArg, HPTGluArg, and HPTAadArg were confirmed by NMR methods. The fraction folded of the peptides was determined using chemical shift data involving the fully unfolded and the fully folded reference peptides. The interaction free energy followed the trend: Aad-Arg > Glu-Arg ≈ AspArg. Apparently, the longer the negatively charged residue side chain length, the stronger the ion pairing interaction. HIV-1 Tat protein contains an arginine-rich sequence (Tat49-57), which binds specifically to the trans-activating responsive (TAR) element and plays an important role in nuclear localization. The binding of HIV-1 Tat protein and TAR RNA is essential for HIV-1 virus genome replication. To study the effect of arginine dimethylation on RNA recognition and cellular uptake, each arginine residue in Tat49-57 was replaced with a dimethylated arginine including ADMA and SDMA, the asymmetric and symmetric dimethylated forms, respectively. The dissociation constant for the Tat derived peptide-TAR RNA complexes was determined by gel shift assay. The cellular uptake efficiency of these Tat derived peptides into Jurkat cells was assessed by flow cytometry.