利用分子動態模擬探討單鏈DNA結合蛋白之C端結構機械性質

Using Molecular Dynamics Simulation to Study the Structural and Mechanical Roles of the Intrinsically Disordered C-terminal Domain in Escherichia coli Single-Stranded DNA-Binding Protein (SSB)

張皓婷(Hao-Ting Chang)

指導教授 ： 朱智瑋

國立交通大學/生物科技學院/分子醫學與生物工程研究所/碩士(2020年)

摘要

大腸桿菌單鏈去氧核糖核酸結合蛋白(EcSSB)與單股去氧核糖核酸(ssDNA)的結合,具有高親和性但無序列專一性。EcSSB與ssDNA的結合可防止結構不穩定之ssDNA產生二級結構而發生核水解。EcSSB在與ssDNA結合的同時,會與許多特定的大腸桿菌單股去氧核糖核酸結合蛋白交互作用蛋白(SIP)在DNA代謝過程中協同合作。EcSSB之N端結構為核苷酸/寡糖結合結構(OBD),其C端結構與結構上定義明確的N端結構不同,為一段無法由結晶結構觀察到之固有無序鏈結分子。C端結構由固有無序鏈結分子(IDL)和九個氨基酸序列的模體(TIP)(MDFDDDIPF)組合而成。先前的研究提出在ssDNA存在的情況下、C端結構TIP與其它EcSSB或其它特定SIP共同參與關鍵之協同作用,C端結構IDL則形成特殊構型、提供 EcSSB蛋白本身保護而免於水解。為了進一步了解EcSSB蛋白內部與外部交互作用之機械性質,本研究使用微秒長度之全原子分子動態模擬進行EcSSB於無ssDNA結合與有 ssDNA結合、及高鹽類濃度與低鹽類濃度環境下之分子動態模擬。此外,分析方法如氫鍵計算、主成分分析(PCA)與機械性質分析用於計算、量化 EcSSB 內部與外部交互作用之結構與力學特性。最後本研究發現C端結構IDL中的極性非帶電性氨基酸(Ser、Gln和Asn)具有多變之內部與外部機械性質彈性。如此高彈性之內部與外部機械性質意味著,這些極性非帶電性氨基酸組合成之多個弱交互作用結合位是EcSSB自我保護或與其它蛋白結合ssDNA並協同作用之關鍵。此外,本研究觀察到EcSSB與ssDNA結合後,C端結構的結構彈性增加;而在低鹽濃度下,C端結構發生向外延展之結構機率也增加。

關鍵字

單鏈DNA結合蛋白 ； 單股DNA ； 固有無序鏈結分子 ； 分子動態模擬

並列摘要

Escherichia coli single-stranded DNA binding protein (EcSSB) binds to the single-stranded DNA (ssDNA) with high affinity but no sequence specificity, preventing ssDNA from nucleolytic degradation while recruiting a group of specific EcSSB interacting proteins (SIPs) that cooperate in the DNA metabolic processes. In contrast to the well-defined N-terminal oligonucleotide/oligosaccharide-binding domain (OBD), the C-terminal domain containing intrinsically disordered linker (IDL) and a nine amino acid motif (TIP) (MDFDDDIPF) can not be observed in any crystal structure. Previous studies suggest that the C-terminal domain plays crucial roles in cooperating with other EcSSB subunits or other specific SIPs, and the structural properties of IDL afford proteolysis protection especially in the presence of ssDNA. To understand the mechanism by which EcSSB cooperates with EcSSB or specific SIPs, μs-timescale all-atom MD simulations of apo EcSSB and ssDNA bound EcSSB are performed. Furthermore, a fluctuation-matching scheme are used to quantify the interactions between residues in EcSSB. The polar residues (Ser, Gln, and Asn) in IDL are found to have heterogeneous coupling strengths in intra-domain interactions and in inter-domain interactions. Such diverse interactions imply that such residues are key for the ability of IDL in interacting with multiple targets. Furthermore, the flexibility of C-terminal domain is observed to increase in the presence of ssDNA, and lower C-terminal domain flexibility is also seen at the low salt concentration.

並列關鍵字

Single-Stranded DNA-Binding Protein ； ssDNA ； Intrinsically Disordered Linker ； Molecular Dynamics Simulation

參考文獻

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