攜鐵蛋白自組裝調控性及其蛋白組胺酸轉烷基酶功能探討

Ferritin engineering for tuning assembled cage and catalyzing protein histidine alkylation

梁桓瑄(Huan-Hsuan Liang)

指導教授 ： 王彥士

國立臺灣大學/生命科學院/生化科學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102707

摘要

攜鐵蛋白為一種自組裝蛋白並廣泛存在於各種生物中，功能為攜帶氧化鐵奈米粒子及具鐵離子氧化還原酶活性。攜鐵蛋白具有良好的生物相容性、耐熱性和內部中空，在拓展攜鐵蛋白功能上，搭配標靶識別及藥物釋放功能，可做為奈米裝載工具以運輸小分子或胜肽藥物。另一方面，攜鐵蛋白球表面含金屬介面也具有發展新型酵素催化反應潛力。本研究旨在以人類重鏈攜鐵蛋白為材料，探討攜鐵蛋白鏈結光敏蛋白自組裝結構在不同酸鹼及光照下的穩定度以及優化本實驗室以此發展的含金屬蛋白質組胺酸轉烷基酶。
在利用酸鹼及光照調控攜鐵蛋白球自組裝解構研究上，利用生物感知藍光造成結構變化的光敏蛋白連接於攜鐵蛋白前端，利用波長480 nm的光源影響攜鐵蛋白單體之間作用力，在L1S-Ftn的自組裝結構上發現對於在pH = 6-5.5會緩步解構，之間對於480 nm光照有立即解構效應。此外，已發展在C2接觸面嵌入非典型胺基酸的含金屬蛋白質組胺酸轉烷基酶，其具有專一性對胰島素的HisB5官能基修飾的功能，透過單一或雙位點的非典型胺基酸取代及不同的銅二價金屬鹽類螯合嘗試，優化針對蛋白受質胰島素及小泛素化蛋白上位置特異的組胺酸進行傅-克烷基化催化反應及產率提升，其中在以Ftn-63/67-2酵素並添加Cu(OAc)2的可催化小泛素化蛋白中單一組胺酸1-異丙二酸基二乙酯-2-異丙烷基化產率達33%。

關鍵字

攜鐵蛋白 ； 光敏蛋白 ； 蛋白質與蛋白質之間的作用力 ； 蛋白質組胺酸轉烷基酶 ； 組胺酸修飾 ； 蛋白質工程

並列摘要

Ferritin is a group of native self-assembly protein, which exist in many kinds of species. The functions of the ferritin are storing iron ion and ferroxidase activity. Because the properties of ferritin are biocompatibility, heat resistance and hollow cavity, it is good for ferritin to engineering its functions. Ferritin has been applied in the study of drug delivery which is a controllable system by releasing cargos or turning off the reaction via chemical conditions changing from environment in the target site.
In controlling the disassembling of ferritin cage, a LOV domain fusion at the N terminal of the ferritin, L1S-Ftn, which is a kind of blue light-activated protein, was constructed and form self-assembled 24mers. Assembled L1S-Ftn protein was found sensitive in lower pH value and resulted in cage dissembling from pH = 6.0 to 5.5. Quick light-driven dissembling was also occurred at pH = 6-5.5 under 480 nm light treatment. In our previous work, human heavy chain ferritin was designed in C2 interface remodeling for copper (II) chelating and demonstrated in synthesizing insulin analogues with site-specific modification on HisB5. In optimizing the ferritin enzyme prototype in modifying protein histidine, one or two sites-directed mutagenesis with non-canonical amino acids at ferritin C2 interface and various copper (II) salts were evaluated using insulin and SUMO1 as protein substrates. Enzyme Ftn-63/67-2 with Cu(OAc)2 catalyzed the Friedel-Crafts alkyl reaction toward one of three histidine residues of SUMO1 protein with diethyl ethylidenemalonate (DEEM) in 33% yield.

並列關鍵字

Ferritin ； Light-oxygen-voltage-sensing domain ； Protein-protein interactions ； Metalloenzyme ； Protein histidine modification

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