精胺酸側鏈長短與胍基對β-Hairpin結構穩定度及核糖核酸辨認之影響

Effect of Arginine Side Chain Length and Guanidinium Group on β-Hairpin Stability and RNA Recognition

張嫺楨(Hsien-Chen Chang)

指導教授 ： 陳平

國立臺灣大學/理學院/化學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10837

摘要

靜電作用力對於穩定蛋白質結構扮演相當重要的角色，主要由帶相反電荷的胺基酸所提供。然而，帶電荷胺基酸雖具有不同側鍊長度之類似物但卻不為自然界生物所利用。為研究側鍊長短對於β-hairpin結構穩定之影響，遂利用固相合成法合成出一系列帶有不同類似物(Agb, Agp及Dap)的胜肽，並以二維核磁共振光譜(TOCSY, ROESY及 DQF-COSY)找出胜肽鏈中各殘基氫原子之化學位移。由各殘基α氫原子化學位移之偏移，3JHNα偶合常數及NOE效應，可確認胜肽分子所形成之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跨股側向作用力之能量大小以雙突變循環分析，結果顯示：離子對Asp-Agb與Asp-Agp有相近的能量，而離子對Asp-Dap較Asp-Agp能量高。側鍊長短對於離子對Arg-Asp作用力沒有影響，而無胍基之胜肽具有較強之離子對作用力。
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。Tat蛋白上47-57位置是兩者專一性結合的區域，此區域上具有六個Arg，對於TAR RNA辨認相當重要。將此六個Arg分別取代成Lys，利用電泳偏移分析Tat衍生物對TAR RNA之結合能力與專一性，結果顯示，將Arg取代成Lys時，Tat與TAR RNA之結合能力降低。而Tat -Lys53在polydIdC或tRNA存在下，專一性較正常態大為降低，顯示Arg 53對於Tat蛋白與TAR RNA之專一性結合影響重大。

關鍵字

靜電作用力 ； 帶電荷胺基酸 ； β-sheet ； β-hairpin ； RNA辨認

並列摘要

Electrostatics is important for protein structure stability. The interaction can form between oppositely charged residues. Interestingly, the natural charged amino acids, Arg, Lys, Glu, and Asp all have analogs with alternative side chain lengths. However, these analogs are not used in natural proteins. In order to study the effect of side chain length on β-hairpin stability, peptides HPTAspXaa, containing different charged amino acid analogs Agb, Agp and Dap, were synthesized by solid phase methods. The sequence specific assignments were based on TOCSY, ROESY, and DQF-COSY spectra. The hairpin structures were confirmed by chemical shift deviation, 3JHNα coupling constants and NOE signals. The fraction folded and delta Gfold of peptides were derived by comparing the chemical shifts with the folded and unfolded reference peptides. The double mutant cycles were applied to analyze the cross strand lateral Asp-Xaa interaction energy. The Asp-Agb and Asp-Agp have similar interactions energy in β-hairpins, suggesting the different side chain lengths of Arg analogs do not affect ion-pairing interactions with Asp. The Asp-Dap pair was more stabilizing than the Asp-Agp pair in β-hairpins, which may be due to the charge dispersion of the guanidinium group on Agp.
HIV Tat protein and TAR RNA binding is an essential step for HIV-1 virus genome replication. A short region (residues 47-57) in the Tat protein binds to the TAR RNA specifically. This region contains six Arg residues, which are important for TAR RNA recognition. The Arg in Tat peptide was individually replaced with Lys to study the effect of the guanidinium group on TAR RNA recognition. The binding affinity of all Tat-derived peptides decreased, when introducing Lys. The KD of Tat-Lys53 increased in the presence of polydIdC and tRNA showing that the guanidinium group at Arg53 is particularly important for RNA recognition.

並列關鍵字

electrostatics ； charged amino-acids ； β-sheet ； β-hairpin ； RNA recognition

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