Development of Polymerized Liposomes for Protein Recognition

智能优点：有人建议开发能够识别特定蛋白质的脂质体，这种蛋白质的表面拓扑已经印在脂质体的表面上。这项研究的目标蛋白是基质金属蛋白酶-9，它参与了许多生理和病理过程。脂质体将使用各种脂类(阳离子、中性和阴离子)构建，以允许调整脂质体，使其凝胶转变温度高于目标蛋白质经历热变性的温度。具有适合光聚合的官能团的脂类将被包括在脂类混合物中。一些脂类还会有专门与酶结合的头部基团，以便相对于脂质体表面唯一地定位蛋白质。在脂质体凝胶转变和蛋白质变性温度之间的温度下孵育复合体期间，膜的流动性将允许脂质体将其表面调节到蛋白质表面，并为以后与蛋白质结合形成补充模板。在脂质体的凝胶转变下方冷却和脂质体膜的光聚合，然后锁定在模板中。初步工作表明，该酶对模板脂质体的亲和力比未模板脂质体提高了1-2个数量级。本工作将如下进行：(I)合成结构多样的可聚合脂类，以形成用于同工酶选择性检测和抑制基质金属蛋白酶-9(MMP9)的脂质体，(Ii)制备具有不同头基的聚合脂质体作为高度特异的多管齐下的基质金属蛋白酶同工酶抑制剂，(Iii)利用螯合的Eu(III)和Tb(Iii)开发基于荧光的传感器，用于检测MMP9的同工酶选择性，(Iv)确定脂体辅助检测和抑制MMP9BROADER影响的机制：本工作为分子印迹开辟了一条新的途径，特别是在蛋白质识别和“人工抗体”制备领域。可以想象在基于脂质体的生物战剂诊断工具、植物和动物病原体的检测以及预防和治疗药物的开发中的应用。PI将支持两名在实验室工作的美国原住民本科生。这两家公司都将在各自的实验室里接待高中生和公立学校教师，参加NDSU赞助的暑期研究项目。

INTELLECTUAL MERIT: It is proposed to develop liposomes that have been designed to recognize specific proteins, the surface topology of which has been imprinted on the surface of the liposomes. The targeted protein for this study is matrix metalloproteinase-9, which is involved in a number of physiological and pathological processes. Liposomes will be constructed using a variety of lipids, cationic, neutral, and anionic, in order to permit tuning of the liposome so that its gel transition temperature lies above the temperature at which the target protein undergoes thermal denaturation. Lipids possessing functionality suitable for photopolymerization will be included in the lipid mixture. Some of the lipids will also have head groups that bind specifically to the enzyme so as to orient the protein uniquely with respect to the liposome surface. During incubation of the complex at a temperature intermediate between the gel transition of the liposome and the denaturation temperature of the protein, the fluidity of the membrane will permit the liposome to accommodate its surface to the surface of the protein and form a complementary template for later binding of that protein. Cooling below the gel transition of the liposome and photopolymerization of the liposome membrane then locks in the template. Preliminary work shows that the affinity of the enzyme for the templated liposome is enhanced by one - two orders of magnitude relative to the untemplated liposome. The work will proceed as follows: (i) synthesize structurally diverse, polymerizable lipids to formulate liposomes for isozyme-selective detection and inhibition of matrix metalloproteinase-9 (MMP-9), (ii) formulate polymerized liposomes with different types of head groups to serve as highly specific multi-prong inhibitors for MMP isozymes, (iii) develop fluorescence based sensors using chelated Eu(III) and Tb(III) for the isozyme-selective detection of MMP-9, (iv) determine the mechanisms of the liposome assisted detection and inhibition of MMP-9BROADER IMPACTS: The proposed work opens a new avenue to molecular imprinting, especially in the area of protein recognition and "artificial antibody" preparation. Applications can be imagined in liposome-based diagnostic tools for biological warfare agents, detection of plant and animal pathogens, and development of prophylactic and therapeutic agents. The PIs will support two Native American undergraduates working in the lab. Both will host high school students and public school teachers in their laboratories in NDSU-sponsored summer research programs.