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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 吳世雄(Shih-Hsiung Wu) | |
dc.contributor.author | Sho-Fine Chang | en |
dc.contributor.author | 張修桓 | zh_TW |
dc.date.accessioned | 2021-06-13T04:24:22Z | - |
dc.date.available | 2007-07-28 | |
dc.date.copyright | 2006-07-28 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-21 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33083 | - |
dc.description.abstract | Tc32 是從一種亞馬遜毒蠍 Tityus cambridgei 的毒液中所純化出來的鉀離子通道抑制胜肽,其全長由 35 個氨基酸所組成 (TGPQT-TCQAA-MCEAG- CKGLG-KSMES-CQGDT-CKCKA) ,含有三對雙硫鍵。在活性分析實驗上,它可以抑制 Shaker/Kv1.x 型式的鉀離子通道。與其他 a-KTxs的序列比較之下,Tc32被歸類在第十八個 a-KTxs的次家族第一個成員 (a-KTx 18.1)。先前,我們實驗室對 Tc32 的三級結構研究顯示,它與一般的 a-KTxs 的結構型態類似,都是雙硫鍵穩定的 a/ b scaffold 構型。但在與其他對於抑制 Shaker/Kv1.x 型鉀離子通道的 a-KTxs 做序列比對之下發現,某些序列恆定且與離子通道有重要作用力的氨基酸在 Tc32 中都沒有出現,這顯示著 Tc32 在抑制 Shaker/Kv1.x 型鉀離子通道的機制似乎有別於傳統。進一步以 docking model 探討 Tc32 與 Kv1.3 鉀離子通道之間的作用,我們發現其中 Lys34 在抑制 Kv1.3 鉀離子通道中扮演著重要的角色,它以帶正電荷的官能基伸入鉀離子通道的 selectivity filter 並與 GYG-motif 骨架中帶部份負電荷的羰基作用。其他如 Gln4、 Lys17、 Leu19、 Lys32 可能負責辨識或輔助的作用。在突變種 K34A 的結構分析上,我們發現 K34A 與 Tc32 的三級結構有高度的相似性,在 N 端會形成一段 a-helix (Ala9-Leu19),在 C 端形成一對反向平行的 b-sheet (Ser22-Gln27 and Thr30-Ala34),並由三對分子內雙硫鍵所鍵結 (Cys7-Cys26, Cys12-Cys31, and Cys16-Cys33)。然而在官能基的分佈和表面電荷圖下,我們推測官能基效應可能造成對 Kv1.3 鉀離子通道抑制性的影響。爾後,在 Tc32 以及其不同的突變種來做一系列的活性分析實驗,相信更能夠確定及加強我們的推測與分析。 | zh_TW |
dc.description.abstract | Tc32 is a K+ channel-blocking peptide purified from the venom of Amazonian scorpion Tityus cambridgei and composed of 35 amino acid residues (TGPQT- TCQAA-MCEAG-CKGLG-KSMES-CQGDT-CKCKA) cross linked with three disulfide bridges. Tc32 is classified as the first number of the subfamily of a-KTx (a-KTx 18.1) based on sequence comparison with other members in a-KTx. In electrophysiological experiment, it shows blocking activity against Shaker/Kv1.x type channel. The three-dimensional structure of Tc32 has been previously investigated by our laboratory, and the structural fold of Tc32, a disulfide-stabilized a/b scaffold, is very similar with other a-KTxs. In sequence alignment analysis, some important residues of Tc32 are different from those in other a-KTxs. This indicates the recognition or blocking mechanism of Tc32 may be different. The docking model of Tc32 with Kv1.3 channel suggests that Lys34 inserts into the selectivity filter and interacts with the backbone carbonyls of GYG-motif from each subunit of the channel. In addition, other residues, such as Gln4, Lys17, Leu19, and Lys32, may be also involved in the recognition or blocking function. Structural studies show that the 3D structure of Tc32 and K34A mutant are very similar and they all contain an a-helix (Ala9-Leu19) and a double strand anti-parallel b-sheet (Ser22-Gln27 and Thr30-Ala34). The conformation is hold by three intramolecular disulfide bridges (Cys7-Cys26, Cys12-Cys31, and Cys16-Cys33). On the basis of the side-chain distribution and the potential surface plot, we suppose that side-chain effect should possess a greater impact on the blocking activity. Further bioassay of Tc32 as well as its mutants may reenforce our assumption. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T04:24:22Z (GMT). No. of bitstreams: 1 ntu-95-R93B46018-1.pdf: 2191516 bytes, checksum: 937ecc3b59b2e282c964b4cca1c511aa (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 中文摘要 ……………………………….……………………………………… 1
Abstract ………………………………………………………………………. 2 Chapter 1: Introduction ..……………………………………………………... 3 1. Ion channels ……………………………………………………………. 3 2. Channel blockers from scorpion toxin ...……………………………… 4 3. Scorpion toxin “Tc32” ………………………………………………... 6 4. Docking of Tc32 to Potassium channel ………………………………. 8 Chapter 2: Materials and Methods …………………………………………. 16 1. Solid phase peptide synthesis ………………………………………… 16 2. Refolding and purification of the crude peptide ……………………… 17 3. Nuclear Magnetic Resonance (NMR) spectroscopy …………………. 18 (a.) Sample preparation and procedures …………………………... 18 (b.) The NMR signals assignment ………………………………… 19 (c.) Structure calculations ………………………………………… 19 (d.) Structural comparison between Tc32 and K34A ……………... 20 Chapter 3: Results …………………………………………………………… 26 1. Prediction of the properties of Tc32 and its analogs …………………. 26 2. Peptide “K34A” synthesis, refolding and purification ……………….. 26 3. Nuclear Magnetic Resonance (NMR) spectroscopy of K34A ………... 27 (a.) Sequential assignment and secondary structure determination of K34A ………………………………………………………… 27 (b.) Solution structure of K34A …………………………………… 28 (c.) Structure comparison of Tc32 and its mutant K34A …………. 29 Chapter 4: Discussion ………………………………………………………... 53 References ……………………………………………………………………. 57 | |
dc.language.iso | en | |
dc.title | 蠍毒中鉀離子通道抑制胜肽 Tc32 突變種之核磁共振結構分析 | zh_TW |
dc.title | Synthesis and structural analysis of mutant K+ channel blocker Tc32, from the Scorpion Tityus cambridgei | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 陳金榜(Chinpan Chen) | |
dc.contributor.oralexamcommittee | 張文章 | |
dc.subject.keyword | 毒蠍,核磁共振,蠍毒,鉀離子通道抑制胜肽, | zh_TW |
dc.subject.keyword | NMR,Scorpion toxin,Tc32,K34A,Tityus cambridgei, | en |
dc.relation.page | 60 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-22 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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