利用支撐式脂質雙層膜平台來研究磷脂酶與Min蛋白和脂質膜間之交互作用

Abstract

支撐式脂質雙層膜 (Supported Lipid Bilayer, SLBs) 為一種常用於生物技術的仿生膜，適用於研究脂質膜和生物分子之間的相互作用與物理特性。脂質雙層膜與基材之間水層，使脂質雙層膜保留了二維流動性的特性，能讓嵌入膜的生物分子之性質與結構都不會受到破壞。在本論文中，我們使用此平台來研究磷脂酶(PLA2) 和Min蛋白在脂質膜上的反應機制。 磷脂酶是種膜週邊蛋白，能水解甘油磷脂在sn-2的位置，並從中釋放出溶血磷酸膽鹼和脂肪酸。目前文獻發現磷脂酶在各種細胞程序中扮演重要的角色，並認為它有潛力被用於藥物輸送，能將藥物有效的釋放到目標區域。我們觀察到磷脂酶的水解反應可誘導脂質膜界面，形成具有沾黏性之生物分子模板，且其形成和脂質膜的流動性有關。我們製備了不同莫爾比例的二棕榈酰磷脂酰胆碱/二油酰磷脂酰胆碱 (DPPC/DOPC) 脂質膜以調控膜的反應與流動性。我們觀察到經磷脂酶誘導形成的沾黏性生物分子模板，只有在特定比例下的之二棕榈酰磷脂酰胆碱/二油酰磷脂酰胆碱 (DPPC/DOPC) 脂質膜中，才有顯著地生成，在純二棕榈酰磷脂酰胆碱 (DPPC) 與純二油酰磷脂酰胆碱 (DOPC) 脂質膜中生成的量卻很少，也因此不容易在以前的研究中被觀察到。標記螢光分子的磷脂酶實驗結果證明了沾黏性生物分子模板確實是由磷脂酶誘導形成的。此外，原子力顯微鏡 (Atomic Force Microscope, AFM) 結果表明生物分子模板具有階梯狀結構，有可能是磷脂酶的水解產物在脂質膜上堆積排列而成。其堆積結構會造成脂質的疏水端會暴露於外部水溶液環境中，導致磷脂酶和其他類型的蛋白質，如蛋白聚糖 (Proteoglycan) 與鏈霉親和素 (Streptavidin)，會沾黏到這些疏水段區域來保護疏水端。這些實驗結果表明，磷脂酶能誘導原屬於兩性離子界面的脂質膜變成一種具有非專一沾黏性之生物分子模板，因而造成其它生物分子的沾黏。 Min蛋白經研究發現能在大腸桿菌細的兩極來回動態振盪，調控細胞分裂的位置。然而，Min蛋白與脂質膜之間是如何交互作用依然未知。為了進一步觀察磷脂酶是如何影響脂質膜，我們製備了有標記螢光分子的二油酰磷脂酰胆碱/二油酰磷脂酰甘油钠盐 (DOPC/ DOPG) 脂質膜以模仿大腸桿菌之細胞膜，以研究Min蛋白和脂質膜間的交互作用。我們推測Min蛋白的振盪可以改變脂質膜中的密度差，因此影響其它嵌入膜中生物分子的動態行為。當標記螢光MinD, 標記螢光MinE, 三磷酸腺苷(Adenosine Triphosphate, ATP)與三磷酸腺苷再生系統 (ATP regenerating system)，被加入脂質膜後，我們能觀察到膜中之鏈霉親和素-生物素-磷脂質复合物 (Streptavidin-biotinylated-DHPE) 會在二油酰磷脂酰胆碱/二油酰磷脂酰甘油钠盐 (DOPC/DOPG) 脂質膜上會產生具有波紋運動型態之濃度分佈。根據膜中之鏈霉親和素-生物素-磷脂質复合物 (Streptavidin-biotinylated-DHPE) 以及MinD 和MinE 濃度分佈的結果，膜中之鏈霉親和素-生物素-磷脂質复合物(Streptavidin-biotinylated-DHPE) 分子並不是直接與MinD與MinE反應，波紋運動的形成很有可能是由Min蛋白在進行振盪時的吸附與解離機制造成的。目前的結果顯示，Min蛋白可能藉由一個類似線性蠕動泵的輸送機制來誘導嵌入脂質膜上的生物分子沿著Min蛋白的移動方向輸送。

Supported lipid bilayers (SLBs) have been developed to provide a well-controlled in vitro platform for studying the biophysical properties and interactions between lipid membranes and biomolecules. The bilayer structure allows the embedded membrane species to maintain their native orientation, and the two dimensional fluidity is a crucial factor for bio-molecular interactions to occur. In this report, we are using SLBs platform to study the properties and mechanism of phospholipase A2 (PLA2) and Min proteins. Phospholipase A2 (PLA2) is a peripheral membrane protein that can hydrolyze phospholipids to produce lysolipids and fatty acids. It has been found to play crucial roles in various cellular processes and thought as a potential candidate for triggering drug release from liposomes for medical treatment. Here, we directly observed that PLA2 hydrolysis reaction can induce the formation of PLA2-binding domains at lipid bilayer interface and found that the formation was significantly influenced by the fluidity of the lipid bilayer. We prepared supported lipid bilayers (SLBs) with various molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to adjust the reactivity and fluidity of the lipid bilayers. A significant amount of the PLA2-induced domains was observed in mixtures of DPPC and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) but not in either pure DPPC or pure DOPC bilayer, which might be the reason that previous studies rarely observed these domains in lipid bilayer systems. The fluorescently labeled PLA2 experiment showed that newly formed domains acted as binding templates for PLA2. The AFM result showed that the induced domain has stepwise plateau structure, suggesting that PLA2 hydrolysis products may align as bilayers and accumulate layer by layer on the support and the hydrophobic acyl chains at the side of the layer structure may be exposed to the outside aqueous environment. The introduced hydrophobic region could have hydrophobic interactions with proteins and therefore can attract the binding of not only PLA2 but also other types of proteins such as proteoglycans and streptavidin. The results suggest that the formation of PLA2-induced domains may convert part of a zwitterionic nonsticky lipid membrane to a site where biomolecules can nonspecifically bind. Min proteins have been shown to dynamically oscillate in E.coli and play important roles in regulating the cell division. However, it is still unclear how Min proteins interact with the lipid membrane. To investigate how the Min proteins influence the lipid membrane, we used fluorescently-labeled DOPC/DOPG supported lipid bilayers (SLBs) to mimic the biological behaviors of the E.coli membrane for studying the Min protein-lipid membrane interaction. We hypothesized that the oscillations of Min proteins could play a crucial role in changing lipid membrane spatial density and therefore influence the dynamics of the other membrane embedded species. We observed that fluorescently Streptavidin-biotinylated-DHPE in the DOPC/DOPG SLBs form traveling waves after the addition of labeled MinD, labeled MinE, ATP and ATP regenerating system (phosphoenolpyruvate and pyruvate kinase). The intensity profile suggests that Streptavidin-biotinylated-DHPE molecules are not directly bound to MinD and MinE proteins, and the wave formation might be due to the transient steric effect caused by the binding and unbinding dynamics of Min proteins. In addition, Min proteins seem to induce the membrane species embedded in the lipid membrane transporting along the Min protein wave moving direction, which is similar to the transport mechanism of a linear peristaltic pump.