三葉型扭結蛋白-YbeA之摺疊機制與功能分析

Abstract

近年來隨著蛋白結構隨著X光晶體繞射以及核磁共振技術的進步，有越來越多的蛋白質結構逐漸被解析與發表，造成蛋白資料庫越來越完整。此外人們發現在自然環境底下，某些蛋白胜肽鏈會自我交錯產生結。在這裡我們以YbeA為對象來研究扭結蛋白摺疊機制，其中YbeA本身為大腸桿菌內的核醣體的核醣核酸甲基化酶，其本身蛋白胜肽鏈會自我交錯產生三葉型扭結，其扭結型式較為簡單，且分子量較小。然而細胞內蛋白質生成的過程本身是一個非常複雜的過程，因此我們希望透過體外重新摺疊的方式來探討扭結的生成。在這裡我們透過圓二色光譜以及螢光光譜儀分析蛋白質的二級結構以及疏水性核心在摺疊過程中的變化。此外我們透過胰蛋白酶分別對YbeA的摺疊態以及未摺疊態反應，依據不同時間點收集產物，然後透過MALDI-TOF分析水解產物序列，結果顯示，其結構中最晚被切下來的部份與pKNOT-環結蛋白質結構資料庫及分析工具所模擬的扭結區域重疊，表示扭結部分在整個蛋白裡頭是最穩定，就算在高濃度尿素環境底下，也無法使其裸露出來被酵素水解。此外我們透過螢光能量共振轉移來分析扭結區位分子間的螢光能量轉移現象，結果顯示不論是在摺疊態或是未摺疊態其扭結區域的距離都沒有改變，根據MALDI-TOF以及螢光能量共振轉移的結果，我們推測YbeA得扭結部分為一個無法打開的結，此外我們透過動力學截流儀結合圓二色光譜來分析YbeA的摺疊機制，結果顯示，不論是在摺疊或是變性的過程的速率都是相當緩慢的，且整個變化都是相當緩慢的，我們推測可能扭結區域穩定蛋白構性變化所造成。

Knotted proteins are more commonly observed in recent years due to the growing number of structures dissolved by x-ray and NMR in the Protein Data Bank(PDB).A small number of natural proteins have knot configurations in its polypeptide backbone. We select YbeA as a model for its small size and simple knot configurations.YbeA is a RNA methyltransferase that contains a deep embedded trefoil knot in its backbone structure. It is a challenge to reveal the knot formation mechanism during the protein biosynthesis process. For exploring those questions,we use trypsin digested matrix-assisted laser desorption/ ionization time of flight (MALDI-TOF) mass spectroscopy technique to analyze the digested intermediates of YbeA indicated the knotted region is the most compact in overall structure, both in unfolded and folded state. That revealed the knotted structure can’t be opened even in high concentration denaturant environment. The similar result was observed by fluorescence resonance energy transfer (FRET) measurement. Meanwhile,folding kinetic of YbeA analyszed by stopped -flow assistted with CD demonstrate it gently changes both in the folding and unfolding process .It reveals that the knot structure may stabilize the protein comformation.