Biomedical Studies and Atomic Force Microscopy

为了扩展我们的生物原子力(Bio-AFM)技术、仪器和数据分析方法，我们在拉曼-AFM仪器方面取得了进展，并启动了新的石英晶体微平衡-耗散(QCM-D)和AFM仪器工作。应用这些扩展技术并共享现有的Bio-AFM设备，我们与NIH内部和外部研究人员合作，推进了一些生物医学研究。取得显著成果的主要分项目包括： (1)我们通过Bio-AFM和相关生物分析，与David Narum博士(NIAID，NIH)、Louis Miller博士(NIAID，NIH)、Patrick Duffy博士和NIH疟疾免疫和疫苗实验室的其他共同研究员合作，研究了五种疟疾候选疫苗的大分子结构和纳米机械特性。疟疾疫苗的这些蛋白质抗原是通过重组蛋白质生物技术生产的，经过提纯，并以适合人体试验和扩大生产的方式进行表征。我们专注于使用AFM、QCM-D和相关研究来了解正在开发的疫苗产品的结构特性，包括最近的REPA-Pfs25结合物和Qbeta病毒样颗粒，在一系列流体和表面条件下。我们的结果对疟疾生物学和人类免疫反应具有重要意义。这一合作促成了两份出版物、许多演示文稿和几份筹备中的手稿。 (2)我们继续与拉尔夫·诺萨尔博士(NICHD，NIH)、艾琳·拉弗教授(Univ.Eileen Lafer)合作，继续我们对笼状蛋白和笼状蛋白包衣小泡(CCV)的AFM研究。德克萨斯健康科学中心，圣安东尼奥)和其他同事。在CCV的亚细胞运输过程中，通过与接合蛋白、膜脂和其他辅因子的相互作用，形成了CCV的外网格笼。这些动态的大分子结构的错综复杂激发了大量的结构和功能研究。我们已经发表了一项研究，通过原子力显微镜以与电子显微镜相当的分辨率来分辨云母表面的三叶虫的不同轮廓。在云母表面和缓冲液下都很容易观察到经典的三足丝状针轮形状，以及非平面的三棱柱构象和三棱柱-三棱柱二聚体。与从牛脑中提纯的天然CCV相似，五角形和六角形晶格结构在带有或不带有AP180接头的各种网状蛋白组件中都得到了很好的显示。我们还在缓冲液中对三棱柱和CCV进行了单分子力谱(SMFS)研究，并首次揭示了三棱柱重链折叠和展开的一系列内部能量势垒，包括七个重复的145aa基序和众多30aa发夹的分子序列和结构周期性。得到了这些区域的动力稳定性。QCM-D技术的加入提高了我们对笼状蛋白及其组装结构的纳米技术表征的准确性。 (3)我们在开发AFM相关纳米技术、表征活细胞的结构和功能、G蛋白偶联受体(GPCR)、结核分枝杆菌膜、细菌生物膜、聚合物和仿生材料以及推进基础表面和材料科学等领域探索了一些额外的壁内和壁外合作。

To expand our biological atomic force (Bio-AFM) technology, instrumentation and data analysis methods, we have made progress with our Raman-AFM Instrumentation and have initiated a new Quartz Crystal Microbalance-Dissipation (QCM-D)and AFM instrumentation effort. Applying these expanding technology and sharing an existing Bio-AFM facility, we have advanced a number of biomedical investigations in collaboration with NIH intramural and extramural researchers. Major sub-projects with notable results include: (1) We have investigated the macromolecular structure and nanomechanical properties of five malaria vaccine candidates via Bio-AFM and related bioanalysis in collaboration with Dr. David Narum (NIAID, NIH), Dr. Louis Miller(NIAID, NIH), Dr. Patrick Duffy and other co-investigators at the Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH). These protein antigens for malaria vaccine are being produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up productions. We have focused on using AFM, QCM-D, and related studies to understand the structural properties of the developing vaccine products, including most recently an rEPA-pfs25 conjugate and Qbeta virus-like particles, under a range of fluid and surface conditions. Our results have implications for malaria biology and human immunological response. This collaboration has contributed to two publications, many presentations, and several manuscripts in preparation. (2) We have continued our AFM studies of clathrin and clathrin coated vesicles (CCVs) in collaboration with Dr. Ralph Nossal (NICHD, NIH), Prof. Eileen Lafer (Univ. Texas Health Sciences Center, San Antonio), and other coworkers. Clathrin triskelia form the outer clathrin lattice cages of the CCVs during subcellular trafficking via interactions with adaptor proteins, membrane lipids, and other cofactors. The intricacies of these dynamic macromolecular constructs have inspired numerous structural and functional studies. We have published a study on resolving variable profiles of triskelia on mica surfaces by AFM at a resolution comparable to electron microscopy. Classical tri-leg, filamentous pin-wheel shapes, as well as non-planar triskelion conformations and triskelion-triskelion dimers, are readily observed both dried on mica surface and under buffers. Pentagonal and hexagonal lattice structures are well visualized in a variety of clathrin assemblies with or without AP180 adaptors, similar to those of the native CCVs purified from bovine brains. We have also produced single molecule force spectroscopy (SMFS) of triskelia and CCVs under buffers and revealed, also for the first time, a series of internal energetic barriers that characterize triskelion heavy chain folding and unfolding, including molecular sequence and structure periodicity for both the seven repeating 145aa motifs and numerous 30aa hairpins. The dynamic stability of these domains has been obtained. The addition of QCM-D technology has enhanced the accuracy of our nanomechnical characterization for clathrin and their assembled structures. (3) We have explored a number of additional intramural and extramural collaborations in such areas as developing AFM-related nanotechnology; characterizing structure and function of live cells, G-protein coupled receptor (GPCR), mycobacterium tuberculosis membrane, bacterial bio-films, polymer and biomimicking materials; and advancing fundamental surface and material sciences.