MyoD蛋白bHLH區塊片段胺端縮減衍生物構型研究

Conformational study of a N-terminal deletion derivative of a fragment in MyoD bHLH region

周義雄(Yi-Shiung Chou)

指導教授 ： 錢偉鈞

朝陽科技大學/理工學院/應用化學系/碩士(2013年)

摘要

MyoD蛋白是一種DNA結合蛋白，其鹼性區塊的螺旋-環-螺旋結構 (bHLH)能與E2A蛋白形成異源二聚體引發DNA分化基因表達。MyoD蛋白與Id1蛋白形成異源二聚體則可抑制DNA分化基因的表達。為了終止Id1蛋白與MyoD蛋白形成異源二聚體抑制分化基因的表達，以MyoD蛋白中bHLH區塊中Helix2為範本設計胜肽Peptide 3C (NH2-Tyr-Ile-Glu-Gly-Leu- Gln-Ala-Leu-Leu-Arg-Asp-Gln-Cys-CONH2)，可與Id1蛋白結合進而阻止Id1蛋白與bHLH家族結合，達到抑制腫瘤細胞增生之效果，自胺端縮減四個胺基酸後之片段與Id蛋白之結合明顯減弱。本實驗以固相胜肽合成法合成縮減胜肽NtD4 (NH2-Leu-Gln-Ala-Leu-Leu-Arg-Asp-Gln-Cys-CONH2)並探討此段胜肽之構型特徵。由圓二色光譜實驗得知，在水中該段胜肽呈現無序纏捲，需在50% TFE中方可被誘導出類似螺旋之構型，根據核磁共振光譜結果顯示，在50% TFE誘導下Ala3~Asp7片段呈現螺旋構型特徵。此一結果顯示即使具有呈現螺旋構型的可能，仍無法與Id1蛋白有效結合，因此Peptide 3C胺端胺基酸應為促進Id1蛋白結合之重要關鍵。

關鍵字

圓二色光譜 ； 核磁共振光譜 ； MyoD蛋白 ； 固相胜肽合成 ； 三氟乙醇

並列摘要

MyoD is a DNA-bindingprotein protein. The HLH motif containing a cluster of amino acid rich in basic residues, could form heterodimer with E2A proteins to activate speficic gene expression. Id proteins act as negative regnlators of transcription factors within the basic helix-loop-helix family. A peptide analog, Peptide 3C derived from a fragment of MyoD was designecl. Peptide 3C showed high affinity for Id1 protein and inhibit the proliferation of several cancer cell.We synthesige an Peptide 3C derivative with deletion in N-terminal fragment (NH2-Leu-Gln-Ala-Leu-Leu-Arg-Asp-Gln-Cys-CONH2) and study the conformation charac tevic of the peptide.The results of circular dichroisn (CD) spectra showed that the peptide existed mainly as a random coil in agueous solution, Introduction of 50% TFE could induce the formation of helical conformation. Base on the experiment results nuclear magnetic resonance (NMR) spectra, under 50% TFE, the helix spanned the fragment of Ala3~Asp7. Our observations suggest that even with the possibility of the formation of helical conformation, it is not sufficient to facilitate the binding to Id1 protein the fragment in the N-terminal of Peptide 3C maybe more aucial to the binding to Id1 protein.

並列關鍵字

nuclear magnetic resonance ； circular dichroisn ； TFE ； solid-phase peptide synthesis ； MyoD

參考文獻

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