利用「螢光相關光譜技術」與「螢光光鉗」研究蛋白質與微脂體的交互作用

Abstract

在碩士班研究期間我建立了兩套實驗系統: 「螢光相關光譜」(FCS, fluorescence correlation spectroscopy) 及「螢光光鉗」(fluorescence optical tweezers)，並將其應用於研究蛋白質與微脂體 (liposomes) 的交互作用。

螢光相關光譜技術主要透過量測微小系統中分子運動造成的自發性螢光強度的擾動來得到分子運動的訊息。因此，所有會造成螢光訊號擾動的物理參數，例如局部的濃度、分子遷移係數 (mobility coefficients) 分子之間或是分子內的特徵反應等，均可以利用螢光相關光譜技術獲得。較特別的是螢光相關光譜技術可應用於熱平衡以及極低濃度的條件下，非常適合應用於生物系統。此外，跟其他的螢光技術比起來，此技術具有非常高的空間和時間解析度。本論文中的第二章內容，即是以FCS研究一個與細胞訊號傳遞相關的蛋白質 (i.e. MARCKS) 與細胞膜間之交互作用以及攜鈣蛋白 (calmodulin) 調控此交互作用。

Myristoylated alanine-rich C kinase substrate (MARCKS) 是生物活體中一種特殊且廣泛分佈的蛋白質。MARCKS透過和細胞膜以及和calmodulin做結合，調控細胞的分泌、膜的運輸、細胞的運動、調節細胞的生理週期。MARCKS跟細胞膜的結合主要是靠靜電交互作用力，利用螢光相關光譜技術我們發現當微脂體中所含之負電性脂質比例增加時，微脂體上所能吸附之MARCKS總量也會隨著增加。在溫度變化的研究中我們也發現，相變化引起的脂質重新集中排列亦可增加微脂體對MARCKS的吸附能力。同時，利用此系統我們也成功的直接觀測到calmodulin與微脂體競爭MARCKS的過程。

上述之MARCKS與細胞膜間的作用，主要控制訊號的傳遞，對於生命體的存所是必需的。然而，有些蛋白質具有細胞毒性，會導致細胞死亡，例如心臟毒素蛋白 (cardiotoxin)。過去的研究發現，心臟毒素蛋白在細胞膜上會以多聚體方式存在，並形成孔洞使得細胞產生洩漏造成細胞死亡。我們對心臟毒素蛋白在細胞膜上形成孔洞及其封閉的動態過程特別感興趣。我們結合光鉗以及螢光偵測系統架設了一組所謂螢光光鉗系統，觀察單一顆微脂體跟心臟毒素蛇毒蛋白的交互作用。我們在微脂體內包埋了螢光分子，並於其溶液中添加了眼鏡蛇心臟毒素蛋白 (Cobra cardiotoxin) 使其在細胞膜上形成孔洞，並利用螢光偵測系統觀察螢光分子從微脂體內之洩露過程。本論文中第三章部分主要是討論此部份之成果。此部分研究仍在進行之中，目前粗步結果顯示，我們已成功建立可鉗住並觀測「單一」微脂體的技術，藉由量測單一微脂體螢光強度的變化，我們也直接觀測到單一微脂體受眼鏡蛇心臟毒素蛋白攻擊後，脂質膜破裂造成洩漏之現象。

During the two-year study, I built and employed two experimental systems: fluorescence correlation spectroscopy (FCS) and fluorescence tweezers to study different aspects of the interaction between proteins and model membrane systems. Specifically, I used FCS to characterize the binding between MARCKS, liposomes, and calmodulin. Besides, I applied a fluorescence optical tweezers system to study the pore-forming and pore-healing of cobra toxins on liposomes.

FCS allows the extraction of information on molecular motion by monitoring the spontaneous fluctuation of fluorescence intensity followed. In general, all processes that cause signal fluctuation are accessible with FCS; diffusion, photobleaching, and chemical reaction are just a few examples. In chapter 2 details my study on MARCKS, liposomes, and calmodulin. A peptide corresponding to the effector domain of MARCKS was synthesized. The association of the MARCKS peptide with liposome and the following dissociation induced by calmodulin are observed with fluorescence correlation measurement. Our results show that: 1) The binding of MARCKSs to liposomes as the ratio of the negative charged lipids increases in the liposomes, 2) MARCKS binds to liposomes more easily due to the phase change of lipids in the liposomes as observed in the heating process, 3) Modulation of MARCKS binding with liposomes by calmodulin was directly observed with FCS.

Cobra cardiotoxin were known to bind with cell membranes, form holes on membranes and eventually lead to the death of cells. I am particularly interested in the dynamics of the pore-forming and hearing on membranes. For this purpose, I built a fluorescence tweezers system and did some preliminary study. The results are included in chapter 3. Single liposomes fluorescent molecules embedded in it were successfully captured and the fluorescence intensity of the single liposome was monitored. At this stage, I have evidence that I have observed the change of fluorescence intensity induced by cobratoxin. The decrease of the fluorescence was explained by leakage of embedded fluorescent molecules due to holes formed by cobratoxin.