Biomedical Studies and Atomic Force Microscopy

随着我们的生物原子力（Bio-AFM）技术，仪器和数据分析方法的进一步扩展，我们与NIH内部和外部研究人员合作，推进了一些生物医学调查。 取得显著成果的主要次级项目包括： (1)我们与大卫·纳鲁姆博士（NIAID，NIH）和由路易斯·米勒博士（NIAID，NIH）领导的疟疾疫苗开发分支的同事合作，通过AFM研究了重组恶性疟原虫裂殖子表面蛋白3（MSP 3）、重组恶性疟原虫环子孢子蛋白（CSP）和其他两种疟疾疫苗候选物的大分子结构。这些用于疟疾疫苗的蛋白质抗原是从大肠杆菌中生产的，经过纯化，并以适合于向人体试验扩大规模的方式进行表征。我们专注于使用AFM和相关的研究，以了解这些抗原的结构特性下的一系列解决方案和微扰条件。我们的研究结果对蛋白质-蛋白质相互作用和人类免疫反应有影响，并为两篇出版物和其他准备中的手稿做出了贡献。 (2)我们继续与Ralph Nossal博士（NICHD，NIH），Eileen Lafer教授（德克萨斯大学健康科学中心，圣安东尼奥）及其同事合作进行网格蛋白和网格蛋白包被囊泡（CCV）的AFM研究。在亚细胞运输过程中，网格蛋白Trifenia通过与衔接蛋白、膜脂质和其他辅因子的相互作用形成CCV的外部网格蛋白晶格笼。这些动态大分子结构的复杂性激发了许多结构和功能研究。 我们已经解决了可变配置文件的triatroia云母表面上的第一次AFM的分辨率可比电子显微镜。经典的三腿，丝状针轮形状，以及非平面的triskelion构象和triskelion-triskelion二聚体，很容易观察到干燥的云母表面和缓冲液下。五边形和六边形晶格结构在具有或不具有AP 180衔接子的各种网格蛋白组装体中很好地可视化，类似于从牛脑纯化的天然CCV的那些。我们还产生了单分子力谱（SMFS）的triazolia和CCV下的缓冲液，并揭示，也是第一次，一系列的内部充满活力的障碍，其特征triazolia重链折叠和展开，包括分子序列和结构周期性的7个重复的145 aa的图案和众多的30 aa发夹。得到了这些区域的动力稳定性。关于这些结果的一份手稿正在审查中，另一份手稿正在编写中。 (3)我们已经探索了一些额外的校内和校外合作者开发和应用进一步的AFM纳米技术等领域的结构和功能的G-蛋白偶联受体（GPCR），结核分枝杆菌膜，活细胞，细菌生物膜，聚合物和仿生材料，和基础表面科学。

With further expansion of our biological atomic force (Bio-AFM) technology, instrumentation and data analysis methods, 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 of a recombinant Plasmodium falciparum Merozoite Surface Protein 3 (MSP3), a recombinant Plasmodium falciparum circumsporozoite protein (CSP), and two other malaria vaccine candidates via AFM in collaboration with Dr. David Narum (NIAID, NIH) and coworkers at Malaria Vaccine Development Branch lead by Dr. Louis Miller (NIAID, NIH). These protein antigens for malaria vaccine are being produced from Escherichia coli, purified, and characterized in a manner suitable for scale-up toward human trials. We have focused on using AFM and related studies to understand the structural properties of these antigens under a range of solution and perturbative conditions. Our results have implications for protein-protein interactions and human immunological response, and have contributed to two publications and other 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 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 resolved variable profiles of triskelia on mica surfaces for the first time 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. A manuscript on these results is under review and another manuscript is in preparation. (3) We have explored with a number of additional intramural and extramural collaborators to develop and apply further AFM nanotechnology in such areas as structure and function of G-protein coupled receptor (GPCR), mycobacterium tuberculosis membrane, live cells, bacterial bio-films, polymer and biomimicking materials, and fundamental surface sciences.