麩胺酸側鏈長短對於螺旋內離子對作用力以及精胺酸側鏈電荷對於辨識核糖核酸與穿透細胞膜的影響

Effect of Glutamate Side Chain Length on Intrahelical Ion Pairing Interaction and Arginine Side Chain Charge on RNA Recognition and Cellular Uptake

翁明暉(Ming-Huei Weng)

指導教授 ： 陳平

國立臺灣大學/理學院/化學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10234

摘要

靜電作用力在穩定蛋白質結構與蛋白質辨識中，是一個重要的作用力。帶電荷胺基酸能夠參與螺旋內離子對作用力，且蛋白質內超過三成的殘基符合α螺旋結構。令人不解的是，自然界中帶電荷胺基酸的側鏈長度皆不一樣。為了探討帶電荷胺基酸側鏈長度對螺旋內離子對作用力的影響，對於可能具有螺旋內麩胺酸-精胺酸和精胺酸-麩胺酸作用力的胜肽做不同側鏈長度及不同殘基間距的研究。胜肽的螺旋性藉由圓偏光二色性光譜儀測得。根據pH 7的數據顯示，胜肽中帶電荷胺基酸側鏈長度越長 (麩胺酸、天門冬胺酸)，具有越高的α螺旋結構。
在人類免疫缺陷病毒中的蛋白質Tat49-57中，具有兩個生物功能: 其一是細胞穿透能力，具有潛力運用到藥物傳遞，其二是可以和TAR核糖核酸結合，是引起後天免疫缺乏症候群關鍵性的一步。至今仍不清楚在Tat49-57中，哪個帶正電荷精胺酸對這兩個生物功能的活性較為重要。因此，將Tat49-57中帶正電荷的精胺酸改成不帶電的瓜胺酸，並對其進行辦識核糖核酸以及細胞穿透的研究。利用膠體電泳位移和螢光各向異性度分析對TAR核糖核酸與Tat胜肽衍生物的解離常數進行研究。利用流式細胞儀評估Tat胜肽衍生物穿透Jurkat細胞膜且進入細胞內部的效率並用螢光顯微鏡觀察。序列中52、53、55號位置的精胺酸對於辦識TAR核糖核酸非常重要。精胺酸側鏈電荷對於細胞穿透的重要性取決於胜肽的濃度。在30微莫耳胜肽濃度下，序列中53、56、57號位置的精胺酸對於細胞穿透的重要性大於
其它位置的精胺酸。57號位置的精胺酸在120微莫耳濃度下對於胜肽細胞穿透的能力影響重大。

關鍵字

固相胜肽合成 ； 電荷胺基酸 ； α螺旋 ； 靜電作用力 ； 辨識核糖核酸 ； 細 胞穿透胜肽

並列摘要

Electrostatic interactions are one of the dominant forces in stabilizing protein structure and in protein recognition. Charged amino acids participate in ion pairing interactions, and more than 30% protein residues adopt an α-helix conformation. It is unclear why the four charged amino acids have the different side chain lengths. Therefore, the effect of charged amino acids side chain lengths on intrahelical ion pairing interactions was investigated. Peptides with potential intrahelical Glu-Arg and Arg-Glu interactions were explored with various side chain lengths and spacings. Helicity for peptides was determined by circular dichroism (CD) spectroscopy. Based on CD spectra at pH 7, the longer side chain length (Glu vs Asp), the more the helicity.
The human immunodeficiency virus (HIV) Tat 9-mer (Tat49-57) has two biological functions: cellular penetration, which may enable drug delivery, and binding to TAR RNA, which is a crucial step in HIV-1 virus proliferation to cause the acquired immune deficiency syndrome. However, it is unclear which positively charged Arg in Tat49-57 is important for the two biological functions. To examine the effect of Arg side chain charge on RNA recognition and on cellular uptake, each positively charged Arg in Tat49-57 was replaced with a neutral citrulline one at a time. The dissociation constant for the TAR RNA-Tat derived peptide complexes was studied by gel shift and fluorescence anisotropy assays. The cellular uptake efficiency for Jurkat cells was assessed by flow cytometry and the peptide-treated Jurkat cells were observed by an inverted fluorescence microscope. The arginines near the middle of the sequence (52, 53, and 55) play important roles for TAR RNA recognition. The importance for Arg side chain charge for cellular uptake depends on the peptide concentration. The positive charge on Arg53, Arg56, and Arg57 are more important compared to the other arginines for penetrating at 30 μM peptide concentration. The positive charge on Arg57 is crucial for penetrating at 120 μM peptide concentration.

並列關鍵字

solid phase peptide synthesis ； charged amino acids ； α-helix ； electrostatic interactions ； RNA recognition ； cell-penetrating peptide

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