透過同源建模、經驗勢能和螢光共振能量轉移來更準確的預測膜蛋白四級結構

Toward a better prediction for the quaternary organization of membrane proteins by using homology modeling, empirical potential and FRET measurements

張簡啓宏(Chang Chien,Chi Hong)

指導教授 ： 楊立威

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2015年)

摘要

得知膜蛋白的三級或四級結構是了解其分子運作機制的重要的一步。
而X-ray繞射結晶法(X-ray crystallography)是一種目前常用於解出膜蛋白結構的技術，然而X-ray繞射結晶法存在純化過程溶解於清潔劑(detergent)時容易失活(denature)和聚合(aggregate)的困難。而結合殘基距離資訊的實驗結果，如螢光共振能量轉移(Fluorescence resonance energy transfer，縮寫為FRET)，電腦3D結構預測提供了一個預測分子三級及四級結構的新管道。本篇論文以CtH+-PPase為研究對象，試圖以FRET的數據，同源建模法(homology modeling)及經驗勢能(empirical potential)推測膜蛋白四級結構。最後分子動力學模擬微調結構顯示CtH+-PPase是一個斜向交叉的穿膜蛋白，兩個單體有著大約29.8度的交角，與SWISSMODEL結構的RMSD為22.6 Å。

關鍵字

分子動態模擬 ； 螢光共振能量轉移

並列摘要

Membrane protein tertiary or quaternary structure study is important for understanding molecular mechanism. X-ray crystallography is one commonly used method for resolving membrane protein structure, however it is easy for proteins to denature or aggregate in the purification process when proteins are solubilized in detergent in this method. With combining experimental residues distance results such as FRET (Fluorescence resonance energy transfer) data, computation 3D structural prediction provides a new way to predict membrane protein tertiary or quaternary structure. Taking CtH+-PPase as a research object in this study, we try to combine homology modeling, empirical potential and FRET data to predict membrane protein quaternary structure. The final molecular dynamics simulation refined structure reveals that CtH+-PPase is a intercrossing membrane protein with 29.8 degree angle between two monomers and RMSD between CtH+-PPase SWISSMODEL and MD result is 22.6 Å.

並列關鍵字

Molecular Dynamics simulations) ； FRET

參考文獻

Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006). The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling. Bioinformatics 22, 195–201.

Chen, R., and Weng, Z. (2002). Docking unbound proteins using shape complementarity, desolvation, and electrostatics. Proteins Struct. Funct. Genet. 47, 281–294.

Huang, Y.-T., Liu, T.-H., Chen, Y.-W., Lee, C.-H., Chen, H.-H., Huang, T.-W., Hsu, S.-H., Lin, S.-M., Pan, Y.-J., Lee, C.-H., et al. (2010a). Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer. J. Biol. Chem. 285, 23655–23664.

Huang, Y.-T., Liu, T.-H., Chen, Y.-W., Lee, C., Chen, H.-H., Huang, T.-W., Hsu, S.-H., Lin, S.-M., Pan, Y.-J., Lee, C., et al. (2010b). Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer. J. Biol. Chem. 285, 23655–23664.

Huang, Y.-T., Liu, T.-H., Lin, S.-M., Chen, Y.-W., Pan, Y.-J., Lee, C.-H., Sun, Y.-J., Tseng, F.-G., and Pan, R.-L. (2013). Squeezing at entrance of proton transport pathway in proton-translocating pyrophosphatase upon substrate binding. J. Biol. Chem. 288, 19312–19320.