標題: 巨觀與單分子分析脂蛋白之摺疊與脂蛋白及脂質之交互作用
Ensemble and single molecule analysis of apolipoproteins folding and apolipoprotein-lipid interaction
作者: 張家靖
CHANG CHIA-CHING
國立交通大學生物科技學系(所)
關鍵字: 蛋白質摺疊;脂蛋白B-100;單分子;極低密度脂蛋白;低密度脂蛋白;protein folding;apoB-100;single molecule;low-density lipoprotein;pre- very-low-density lipoprotein
公開日期: 2013
摘要: 近來研究指出蛋白質與生物膜之結合與細胞膜之表面屈率變化息息相關,或許可藉 此調節生物體生物膜之運送與融合等作用。因此蛋白質與脂質之交互作用在基礎研究 與應用方面均極為重要。然而系統性的分析單分子層次脂蛋白與脂質之交互作用研究 至今闕如。因此單分子層次研究平台之開發以瞭解脂蛋白與脂質之結合及脂蛋白 (例 如VLDL 合成之重要蛋白質,apoB100 與apoC3) 等之製備亦為當前研究所亟需。 Apolipoprotein (apo) B100 為非常低密度脂蛋白(VLDL)與低密度脂蛋白(LDL)之核 心骨架脂蛋白,由於其分子極大且非常疏水性,因此欲研究此一蛋白質之摺疊為一極 具挑戰性之工作。ApoC3 亦為與VLDL 合成之重要蛋白質,但其摺疊機制與apoB100 之關聯仍未確立。藉由本計畫所採用之TIRF-FRET-AFM (total internal reflection fluorescence microscope coupled with florescence resonance energy transfer and atomic force microscope)之先進光學與單分子操作系統量測,則此二蛋白質之單分子摺疊/變 性、蛋白質間之交互作用及蛋白質與不同曲率脂質體之作用關係可得,並可與巨觀動 態摺疊實驗相印證。 因此本研究之成功將不僅可增進我們對脂蛋白摺疊之瞭解,亦可瞭解VLDL 形成時 脂蛋白與脂質之交互作用機制。
Unraveling molecular mechanisms that regulate protein/lipid interaction has emerged as primary research target in both basic and applied science arena. It is known that conformation of membrane binding protein can sense topological changes of membranes (e.g. curvature) in regulating membrane dynamics and trafficking events, such as vesicle budding, tethering, fusion, and fission. However, a systematic analysis of the apolipoproteins/lipid interactions at single-molecule scale is currently unavailable. It is thus imperative to develop a research platform through which the mechanisms of apolipoprotein/lipid assembly (very low density lipoproteins or VLDL formation). Meanwhile, the functional apolipoproteins, such as apoB100 and apoC3 for VLDL formation, isolation are highly desired, too . Apolipoprotein (apo) B100 is the most important structural apolipoprotein for VLDL and other lipoproteins (e.g. LDL or low density lipoproteins). Because of the enormous size (4536 amino acids) and extreme hydrophobicity, it has been challenging to study the folding/reconstitution events of apoB100-containing lipoproteins. ApoC3 is a small (79 amino acids) apolipoprotein that promotes lipid recruitment during VLDL assembly. It remains unclear the folding process/mechanism of apoC3 and interactions between apoB100 and apoC3. By combining TIRF-FRET-AFM (total internal reflection fluorescence microscope coupled with florescence resonance energy transfer and atomic force microscope) system to quantify the rigidity (unfolding) of apolipoproteins, inter-apolipoprotein interactions and interactions between apolipoprotein and model lipid systems, and the unfolding/refolding process of a single apoB100/apoC3 molecule can be described. These results will be compared with ensemble kinetic folding study of apoB100/apoC3 to reveal the lipid-protein interactions during the folding process. Thus, the outcome of the current study will not only gain fundamental knowledge concerning protein folding but apolipoprotein/lipid interaction during VLDL assembly.
官方說明文件#: NSC100-2112-M009-004-MY3
URI: http://hdl.handle.net/11536/95844
https://www.grb.gov.tw/search/planDetail?id=2866682&docId=408087
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