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  • 學位論文

D型肝炎病毒抗原N端區的結構與其核酸結合性質之研究

Studies of structure and nucleic acid binding properties of the N-terminal domain of hepatitis delta antigen

指導教授 : 吳惠南
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摘要


含D型肝炎病毒抗原N端第1至第88個氨基酸的蛋白稱為NdAg。NdAg含極多鹼性氨基酸,其 具極強核酸結合力。NdAg具leucine zipper-like 序列,其極有可能形成coiled-coil結 構。NdAg亦具核糖核酸監護子的活性,其可協助各式核糖核酸摺疊成新的構型。本研究擬 對NdAg的結構以及NdAg與和核酸交互作用後蛋白或核酸分子結構之變化作深入之探討,以 期了解NdAg蛋白之結構與功能。本研究發現NdAg蛋白中D型肝炎病毒抗原第1至第13個氨基 酸序列被刪除、第1至第23個氨基酸序列被刪除或leucine zipper-like 序列內氨基酸發 生插入或置換突變,均使NdAg蛋白的結構發生變化或令NdAg蛋白喪失其?-螺旋結構。然 而這些突變蛋白仍具核酸結合能力及核酸監護子活性。含D型肝炎病毒抗原第24至第75個 氨基酸序列之蛋白即N7蛋白雖不具a-螺旋結構,但當N7蛋白與核酸交互作用後,a-螺旋結 構可被誘發。ac2450am被核酸誘發出a-螺旋結構的能力較N7蛋白差,故D型肝炎病毒抗原 的第50至第75個氨基酸對於NdAg蛋白與核酸分子之交互作用應該十分重要。由CD光譜之變 化及波長260 nm吸光值之增強可知,NdAg蛋白可改變核酸分子之結構,我們並以電子顯微 鏡觀察到NdAg蛋白可使雙股核酸分子產生bubble或枝鏈,此外NdAg蛋白亦可讓supercoile d form之質體轉型為relaxed form。上述這些性質可能與NdAg蛋白之核酸監護子的功能有 關。

並列摘要


NdAg is a fusion protein containing the first 88 amino acids of hepatitis delt a antigen. NdAg is rich is basic amino acids, it binds nucleic acid molecules with broad specificity. NdAg contains leucine-zipper like sequence that may be involved in the formation of coiled-coil. NdAg is an RNA chaperone, it pro motes the unfolding and refolding of RNA molecules. The aim of this thesis is to investigate the structure and function relationship of NdAg. The deletion of the first 13 amino acids or the first 23 amino acids of hepatitis delta an tigen from NdAg together with the introduction of insertion or substitution mu tation(s) to the leucine-zipper like sequence of NdAg destabilized or abolishe d the a-helical structure of NdAg. Nevertheless, these alterations had small effect on the nucleic acid binding and RNA chaperone activities of NdAg. Trun cated mutant N7 and peptide ac2450am that contained amino acids #24-75 and ami no acids #24-50 of hepatitis delta antigen, respectively, did not have stable a-helical structure. The a-helical structure of both N7 and ac2450am could be induced in the presence of nucleic acid molecules. The nucleic acid binding activity of N7 was stronger than that of ac2450am, therefore amino acids #51-7 5 of hepatitis delta antigen seems to be important for the protein-nucleic aci d interaction. The circular dichroism spectroscopy and UV absorbency studies illustrated that NdAg altered the structure of nucleic acid molecules. In add ition, the electron microscope examination indicated that NdAg not only relaxe d the supercoiled plasmid DNA but also melted double-stranded nucleic acids. These properties of NdAg may be important to the RNA chaperone activity of hep atitis delta antigen.

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