2017-08-012024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/695246摘要：> 前言 蛋白在生物系統中扮演各項流程的推手，其序列決定了蛋白的結構與功能。如對蛋白序列與功能的連結進行全面的解析 (序列空間) 將有助於暸解蛋白的生化性質、預測其功能和演化方向、及製作具新功能的合成蛋白。然而探索蛋白序列空間的實驗規模龐大，以長度為N的蛋白為例，研究者須合成並量測20N序列變異株。 > 研究方法 為克服此困境，我將採獨創的高通量技術研究MerR蛋白家族的序列空間。MerR蛋白為一群偵測細菌胞內金屬濃度並啟動排毒基因的轉錄因子，MerR 蛋白在結構上極為相似，但其金屬結合域的序列變異決定各同源蛋白對金屬的選擇性，由於此結合域很短，我可一次合成帶有萬種序列之金屬結合域的MerR蛋白組庫，並量測這些序列變異株對不同金屬的反應。此實驗平台的技術為: (1) 將MerR蛋白活化基因的程度量化為細胞的螢光蛋白表現; (2) 以分選儀收集螢光蛋白表現量不同的細胞; (3) 定序分析各分選群中MerR金屬結合域的組成。 > 預期結果 依上述方法建構的MerR序列空間將幫助我們: (1) 解開蛋白區分金屬種類的分子機制; (2) 提高註釋MerR同源蛋白功能的正確率; (3) 預測MerR蛋白功能演化的路徑。此外，本研究可能發現具高度金屬專一性及靈敏度的MerR變異株，此素材將可用來發展偵測重金屬污染的生物感測器及微電極晶片。 <br> Abstract: > Background Proteins carry out myriads of cellular tasks whose structure and function are dictated by their amino acid composition. A comprehensive understanding of the protein sequence-function correspondence (i.e. sequence space) shall yield critical biochemical and evolutionary insights to guide protein engineering. However, such analysis demands generation and quantification of 20N protein variants for a sequence of length N. > Approach To overcome this numerical challenge, I will take a high-throughput approach to characterize the sequence space of the MerR family. MerR proteins are transcription factors responsible for sensing intracellular metals and activating metal-detoxifying genes in bacteria. Members of the MerR family are structurally conserved but exhibit distinct metal specificity due to variation in their metal-binding domains (MBD). Because this domain consists of merely 7~9 amino acids, we are able to generate a cell-based MerR variant library encompassing tens of thousands of MBD compositions and profile their metal selectivity based on: (1) GFP to report metal-induced transcription; (2) fluorescence-activated cell sorting to collect cells with different GFP levels; (3) deep sequencing to unravel the composition of MBD variants. > Expected Results The aforementioned approach will produce high-resolution sequence-function maps of the MerR MBD under the induction of different metal species. This unprecedented dataset will allow us to: (1) probe the molecular basis of metal discrimination; (2) assist functional annotation of uncharacterized MerR homologs; (3) infer the evolutionary trajectories of MerR proteins in the sequence space. Moreover, the proposed project will likely discover MBD variants with superior metal-binding specificity and sensitivity. These variants will be promising materials for developing biosensors and microelectrode array chips to detecting heavy metal pollution in the environment.蛋白演化生物感測器序列空間金屬結合域轉譯因子多配體對應性protein evolutionbiosensorsequence spacemetal-binding domaintranscription factorligand promiscuity