蛋白質熱穩定性驗證研究

Abstract

由過去的研究結果得知蛋白質熱穩定性是由多種複雜的因子所造成，我們已成功的發 展出單一的量化指標來呈現多種因子的交互關係，並藉此由蛋白質的序列分析來預測 其熱穩定性。因此目前我們計畫將蛋白質熱穩定性的研究進展到實驗驗證的階段，並 利用取得到的實驗資料改進統計預測系統。 我們將建立一個提高蛋白質熱穩定性點突變位置之預測系統，利用過去所建立的嗜熱 蛋白抗熱關鍵的對偶特徵(coupling pattern)資料，來預測蛋白質熱不穩定的序列區塊， 作為後續進行點突變實驗的候選區域。由於不同功能的蛋白質之間親緣關係差異甚 大，在沒有高溫同源序列可參照的情況下，需先建立可適用於非同源關係下的獨立熱 穩定性特徵分析系統，以符合各種狀況的需求。蛋白質對偶特徵可以在沒有親緣關係 的狀況下，分析出蛋白質的熱穩定性。分析所需的來源資料以微生物Archaea(古生菌) 和Bacteria(細菌)等為主。 為兼顧產業發展需求，計畫挑選纖維素分解酵素作為模式蛋白進行點突變實驗，驗證 其熱穩定性確效。並選擇同源酵素中親源關係較低的序列部分，在不影響活性的情況 之下，進行點突變實驗並驗證其熱穩定性效果。預期理論與實驗跨領域的整合研究將 可對蛋白質的熱穩定性預測有重大的突破。

Protein thermostability is affected by multiple factors simultaneously. I had developed some methods to integrate the multiple factors to one scoring function and enable the protein sequences easier to evaluate for the stability change of mutations in single score approach. Therefore, I hope to push this research topic with real experimental evidences, and integrate my experimental data to my statistical prediction system. Our motivation is to complete the web server tool that suggest appropriate mutation site and changing amino acid types while keep the sequence information, and real experiment will be performed to evaluate this tool. According to coupling pattern approach, we will develop a web-based service tool for thermostability prediction. In this tool, user can select unstable local sequence regions and suggest more stable sequences in unstable regions. Due to the fact that homology is a huge distance in different protein families, enhancing protein thermostability is difficult without thermophilic family information. Users need the independent method to predict protein thermostability enhancement without homology data. Coupling pattern approach is designed to apply in protein thermostability prediction without thermophilic family information. The analysis data in coupling pattern approach is from archaea and bacteria proteomes. At the same time, we also select appropriate model enzyme, such as cellulase, for evaluating this prediction tool. This work approach is beneficial to bio-enzyme industrial development. The proposed mutation site is to select non-homolog region in cellulase protein family. The research motivation is to enhance thermostability and less focus on the enzyme activities.