麩胺酸側鏈長度對α-helix的影響及精胺酸側鏈電荷對辨認DNA的影響

Abstract

蛋白質超過30 % 結構含有α螺旋結構．螺旋內離子對作用力可以穩定結構．側鏈長度與正負電荷相對間距會影響螺旋程度．為了研究側鏈長度對於影響程度的影響，利用固向胜肽合成技術合成並設計一系列含有合成出與精氨酸長度不同的非自然界胺基酸S-2-amino-6-guanidinohexanoic acid (Agh), S-2-amino-4-guanidino-butyric acid (Agb), S-2-amino-3-guanidinopropionic acid (Agp)以及與穀胺酸長度不同的天門冬胺酸的胜肽序列，包括AghAsp3, AghGlu3, AspAgp5, GluAgp5, AgbAsp4以及 AgbGlu4. 利用圓二色光譜儀測量不同胜肽在pH 2-12範圍下的螺旋程度，並且將測量結果根據修飾過的 Lifson-Roig 理論下利用nesting block方法計算出穩定能量．圓二色光譜儀測得訊號結果包括個別胺基酸本身對於螺旋喜好程度、胺基酸序列中側鏈之間與的作用力以及胺基酸側鏈與螺旋骨架N端C端作用力．本研究中，根據圓二色光譜儀訊號顯示pH 7情況下，螺旋程度大小依序為AghGlu3 > AghAsp3, GluAgp5 > AspAgp5, AgbGlu4 > AgbAsp4. 當負電荷胺基酸側鏈越長，其表現出的螺旋程度也相對較高．除外，本研究還針對真核生物轉錄活化蛋白質GCN4 其上面基本區域 (basic region)的精氨酸側鏈電荷來探討GCN4上個別精氨酸對於cAMP response element (CRE) DNA辨識能力的影響．本研究利用瓜氨酸取代精氨酸來探討電荷對於結合能力及選擇性影響，並且利用電泳技術分析合成之胜肽對於特定cAMP response element (CRE) 之間作用關係．本研究顯示WtArgGCN4ss和 MinArg11Ass 作為對照組與其不同實驗組對照發現，WtArgGCN4ss 實驗組顯示相對MinArg11Ass較高的結合能力．除外，WtCit18GCN4ss表現出相對於對照組以及其他實驗組較差的專一性； Min11ACit2ss表現出相對於對照組 (MinArg11Ass) 以及其他實驗組較好的專一性． 本研究亦嘗試利用理論計算的方式模擬合成出的實驗組胜肽與CRE之間的結合情形，並計算出相對應之能量．

More than 30% of protein residues adopt an α-helical conformation. Intrahelical ion-pairing interactions can stabilize the helix conformation. To investigate how side chain length (of charged residues) and relative spacing (between charged residues) affect helicity, peptides AghAsp3, AghGlu3, AspAgp5, GluAgp5, AgbAsp4, and AgbGlu4 were synthesized. The helical content of the peptides were determined by circular dichorism spectroscopy (CD). The helical content of peptides at pH 7 follows the trend AghGlu3 > AghAsp3, GluAgp5 > AspAgp5, AgbGlu4 > AgbAsp4. This trend is consistent with the helix propensity trend Glu > Asp. Peptides with modified sequences of GCN4 basic region were synthesized to investigate the effects of Arg side chain charge on DNA recognition. Peptides WtArgGCN4ss as well as MinArg11Ass, and the mutants with individual Arg residues replaced with citrulline (Cit) at various positions were synthesized. The binding affinity and specificity of the GCN4-based peptides for cAMP response element (CRE) were determined by gel shift assays. The WtArgGCN4ss mutants showed higher affinity than the MinArg11Ass mutants. Further, WtCit18GCN4ss exhibited lower specificity and affinity than WtArgGCN4ss and other WtArgGCN4ss mutants. Min11ACit2ss showed highest affinity and specificity compared to MinArg11Ass and other MinArg11Ass mutants. The results were further corroborated by molecular mechanics calculations.