本研究利用恆溫滴定微卡計(Isothermal Titration Calorimetry, ITC)與圓二色光譜儀(Circular Dichroism, CD)來探討鏈酶卵白素(Streptavidin, SA)與其核適體(aptamer)之間的作用行為。我們藉由調控SA與其核適體(St-1)結合之環境條件，包含在不同金屬離子種類、鹽濃度及溫度下，期望以ITC獲得熱力學資訊，並結合CD觀測二級結構之變化資訊，探討SA與其核適體(St-1)之間的辨識作用機制。 在熱力學的分析，SA與其核適體(St-1)結合反應的發生是由焓主導，表示此結合反應由靜電作用力主導。藉由鹽類的添加，希望能導致靜電遮蔽的效果，證明SA與St-1之結合反應確實為靜電作用力主導，但從ITC之結果發現，隨著鹽濃度的上升，SA與St-1的結合能力不僅未降低，而且SA與St-1結合之放熱量(焓)是增加的，此結果是與預期的結果相衝突的，因此吾人推測SA與St-1結合過程中，在不同鹽濃度下，應有伴隨著不同程度的SA之構型變化所導致的結果。所以利用CD去分析SA與St-1在結合過程中之二級結構的變化，從實驗結果是SA與St-1在結合過程中是有明顯伴隨著構型的改變，但是構型改變量卻不隨著鹽濃度的添加而有所改變。另外，從SA與St-1結合之熱容量變化(?Cp)之分析，顯示SA與St-1在結合過程中，其三級/四級結構變化量是隨著鹽濃度的添加而明顯增加，表示會造成SA與St-1的結合在不同鹽濃度下會有不同程度的整體構型變化，是由於環境中的水分子的排開程度扮演著很重要的關鍵。本研究還利用CD能觀測二級結構之特點，選擇在特定的波長下，測量不同濃度比之SA與St-1的二級結構變化程度，探討結構變化量與化學劑量比(stoichiometry, N)的關係，結果指出一個SA分子最多能結合兩個核適體分子，此結果與ITC之化學劑量比(N=2)相符。從以上的實驗結果，我們提出一個SA與St-1結合的模型，詳細地解釋兩者之間的辨識行為。更進一步地，藉由SA與St-1之結合機制的了解，本研究利用取代滴定法於ITC實驗，克服了ITC之偵測極限，並得到SA與biotin之結合常數。Aptamers are valuable for the discovery and application of new principals and designs of nucleic acid-ligand interactions. This study investigated the thermodynamics and conformational changes associated with the binding between streptavidin (SA) and its DNA aptamer under various temperatures and salt concentrations. The binding was enthalpy-driven with a large entropy cost. The binding association constant (Ka) was independent of the salt concentrations; however, enthalpy increased in conjunction with the salt concentration. The spectroscopic studies indicated that each SA tetramer bound a maximum of 2 aptamer molecules. The binding was accompanied with substantial conformational changes, which were insensitive to the variation of salt concentrations. These non-classical results indicate the prominent involvement of the binding-site hydration water molecules in SA-aptamer binding. We propose a salt-bridge swap model to explain the salt-independent Kd values. To maximize binding affinity, the electrostatic interactions within the proteins and those within the aptamer-Na+ counter ions exchange their partners, resulting in the observed structural changes and the possible release of hydration water molecules.
