硫磺菌乙醯轉移酶抗熱特性之分子動力模擬研究

Molecular Dynamic Simulation Studies on Thermostability Properties of Sulfolobus solfataricus Nα-acetyltransferase (SsArd1)

洪柊羽(Sora Hagawa)

指導教授 ： 溫進德

共同指導教授 ： 徐駿森(Chun-Hua Hsu)

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703214

摘要

硫磺菌Sulfolobus solfataricus之N-乙醯轉移酶(SsArd1)對蛋白質之N端乙醯基修飾對於包括細胞或分子間訊號傳遞等生理功能極為重要，相對於其他物種之同源N-乙醯轉移酶，SsArd1 具有相當高的耐熱特性。SsArd1 構型與人、酵母菌之N-乙醯轉移酶類似，然而SsArd1 具有額外、演化上不保守之環狀結構。一般而言，具有額外環狀結構之蛋白質相較其同源其他蛋白質較不抗熱，因此此研究著重在SsArd1所具有之額外環狀結構如何使SsArd1相對其他同源N-乙醯轉移酶更能抗熱。
由於在高溫下進行蛋白質之結構解析常有實驗上的困難點，故透過取得X光繞射實驗解析出來的蛋白質結構，透過電腦大量計算蛋白質、水分子之所有原子間L-J位能、溫度、動能轉移之間物理關係，透過蒙地卡羅演算法可以得到蛋白質在特定溫度、壓力等條件下隨時間變化最有可能之構型。本實驗採用RCSB PDB資料庫之蛋白結構(PDB: 4R3K)，以Gromacs軟體執行運算。試圖透過結構分析與蛋白質-水分子之交互關係，探討1. SsArd1在不同溫度下本身的穩固程度 (RMSD/RMSF)。 2. SsArd1 在蛋白質加熱變性過程中結構變化及抗熱關鍵結構。 3. SsArd1所具有之額外環狀結構對於蛋白整體二級結構的影響，以及去除額外環狀結構之分子動力模擬變化。 4. SsArd1 酵素催化關鍵部位在高溫下與水分子的交互關係。 5. SsArd1 酵素催化關鍵部位點突變後在各溫度下與水分子作用程度的變化。
透過探討上述問題，SsArd1 之額外環狀結構增加其在高溫時二級結構β摺疊數量，使的SsArd1 在高溫時反而比其他同源N-乙醯轉移酶更穩定，且其酵素催化關鍵部位兩個胺基酸互相穩定機構對於穩定催化關鍵部位、水分子與受質間空間關係極為重要。

關鍵字

硫磺菌乙醯轉移酶抗 ； 抗熱 ； 分子動力模擬

並列摘要

Nα-acetyltransferase (Nat) modifies N-terminal of substrate resulting in critical functions such as signaling for protein degradation, translocation or even apoptosis. Comparing to many Nat homologs, Sulfolobus solfataricus Nα-acetyltransferase (SsArd1) has an extra non-conserved loop. In many studies, longer loop decrease thermostability, however, comparing to homologs, SsArd1 has better thermostability.
Due to difficulties resolving protein structure at high temperature lab experiments, this study went with molecular dynamic simulation approach, calculating conformation based on L-J potentials, kinetic energy transfer and Monte Carlo algorithm using X-ray structure from RCSB PCB database 4R3K and open source Gromacs program. This study focused on 1. Rigidity of SsArd1 at different temperatures. 2. Denature pathway of SsArd1. 3. Relationship between extra loop and SsArd1 secondary structure. 4. Interaction between water and catalytic residues at different temperatures. 5. Impact on modification of catalytic residues.
Through MD simulation results, the extra non-conserved loop become parts of β-sheets, contributing to thermostability. Two catalytic residues stabilize each other, which may be critical maintaining interaction between water-substrate-residues relation.

並列關鍵字

SsArd1 ； Molecular Dynamic Simulation ； Thermostability

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