中文摘要
現今已有大約700 種的扭結蛋白(knotted protein)被確認於蛋白質
資料庫 (protein data bank)，但對於扭結機制的形成以及在蛋白結構中
所扮演的角色仍然不明確。本實驗致力於研究扭結蛋白折疊的機制而
使用一個來自於胃幽門桿菌(Helicolbacter pylori)的蛋白，HP0242。此
蛋白在形成同類二聚體時採用了兩個單體以穿越彼此的方式形成新
穎的偽扭結結構，而這種特性使得HP0242 成為了研究扭結蛋白摺疊
機制一個相當好的模板。
本論文使用各種不同的光學儀器來以不同物理特性分析HP0242
的摺疊機制。我們採用了包含內部以及同步輻射光源的遠紫外線圓二
色光譜(far-UV circular dichroism)確立了此蛋白在化學變性的過程中
有著中間產物的高度分布。內源螢光光譜(intrinsic fluorescence) 只偵
測到自然狀態到達中間產物的過渡時期，原因在於HP0242 只存在一
個色胺酸在靠近蛋白N 端的位置，而色胺酸在完全暴露於溶劑當中
時此蛋白的中間產物仍然有明顯的摺疊部分。因此我們設計了兩個將
苯丙胺酸和色胺酸相互轉換的突變體以便觀察HP0242 其他區域的折
疊情形。綜合了利用小角度X 光散射(small angle X-ray scattering) 以
及動態光散射(dynamic light scattering)所分別觀測到的迴轉半徑
(radius of gyration)以及水化半徑(radius of hydration)我們確立了
6
HP0242 在化學變性的過程中連續的摺疊機制。如今許多的理論模擬
研究致力於扭結蛋白的摺疊機制的分析，而我們對於HP0242 摺疊機
制的分析給予模擬研究提供了關鍵的實驗數據。

Abstract
To date, about 700 knotted proteins have been identified in the protein data bank
(PDB). How and why these protein adopt knotted backbone topologies remain elusive.
HP0242 is a hypothetical protein from Helicolbacter pylori which is homodimeric
and contains a novel interwined topology (a pseudo trefoil 31 knot); it will be properly
knotted when two monomers are concatenated. HP0242 is an ideal model system for
studying knotted protein folding. While far-UV circular dichroism (CD) spectroscopy
with both in-house and synchrotron radiation light sources identifies the existence of a
highly populated folding intermediate during chemical denaturation, intrinsic
fluorescence spectroscopy only probes the native (N) to intermediate (I) state
transition because HP0242 only has one tryptophan residue and it is likely that the
local structure around the tryptophan side chain becomes fully solvent exposed while
a significant part of the structure remains folded in the intermediate state. We
therefore design two Phe-to-Trp mutants to monitor folding events at the other sites of
HP0242. Together with small angle X-ray scattering (SAXS) and dynamic light
scattering (DLS), which report on the changes of radius of gyration (Rg) and radius of
hydration (Rh), respectively, as a function of denaturant concentration, we have
established the sequential folding processes of HP0242 during chemical denaturation.
6
In light the of the emerging interests in employing theoretical modeling approaches to
delineate the folding pathways of knotted proteins, our results provide key
experimental evidence of sequential folding events of HP0242 which can be validated
in the theoretical models.

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