Biomedical Studies and Atomic Force Microscopy

生物膜，蛋白质，亚细胞器，和其他样品正在通过原子力显微镜（AFM），拉曼和荧光光谱，以及相关的生物物理方法，在这个项目涵盖了几个研究合作。 随着我们的技术，仪器和数据分析方法的进一步扩展，我们已经与许多NIH内部和外部研究人员合作，推进了一些这些应用程序。 主要合作包括： (1)我们完成了对结核分枝杆菌衍生的脂质，阿拉伯甘露聚糖脂的作用的研究， (LAM)生物膜的研究。Hayakawa，F.德增，J.A. Dvorak（NIAID/NIH）和同事。 脂阿拉伯甘露聚糖是抗酸杆菌结核分枝杆菌（MTB）细胞壁的组成部分。 结核感染的一个标志是细菌能够破坏宿主免疫反应的正常巨噬细胞防御机制。 据报道，LAM参与抑制这种防御机制的吞噬-溶酶体融合步骤。 我们已经在体外生产了由LAM和不同脂质组成的膜来模拟吞噬体脂质膜，并使用AFM和相关研究量化LAM的影响。 我们发现LAM显著改变膜纳米结构并抑制大的单层囊泡融合，这可能对MTB的存活和人类结核病的战斗具有影响。 (2)我们与大卫·纳鲁姆博士（NIAID，NIH）及其同事合作，通过AFM研究了重组恶性疟原虫裂殖子表面蛋白3（MSP 3）和最近的恶性疟原虫环子孢子蛋白（CSP）的大分子结构。 MSP 3和CSP是人类疟疾疫苗的潜在成分。 这些蛋白质抗原是从大肠杆菌中生产的，经过纯化，并以适合于人体试验规模扩大的方式进行表征。 我们专注于使用原子力显微镜和相关的研究，以了解这些抗原的结构特性的范围内的解决方案的条件下，发现主要是EcCSP单体，但EcMSP 3单体，二聚体，和多聚体的二级结构域的波动折叠。 这种结构应该对蛋白质-蛋白质相互作用和人类免疫反应有影响。 (3)我们与Ralph Nossal博士（NICHD，NIH），Eileen Lafer（德克萨斯大学健康科学中心，圣安东尼奥）及其同事合作，扩大了我们对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。 在这里，我们继续开发和应用新的原子力显微镜（AFM）和相关的分析方案，对triangulia及其组件的表征。 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，以及非平面三聚体的构象和二聚体，很容易观察到干燥的云母表面上，并显示形状的灵活性下缓冲。 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。 我们已经发现了一个相当大的变化，在三氟甲磺酸离子构象和网格蛋白笼纳米力学。我们还制作了缓冲液下的三氧化二氮和CCV的单分子力谱（SMFS），并首次揭示了一系列内部能量屏障， 表征三聚氰胺离子重链折叠和展开，包括7个重复的145 aa基序和许多30 aa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。 (4)我们继续与Shui-Lin Niu博士，Drake Mitchell，Klaus Gawrisch（NIAAA，NIH）及其同事合作，使用AFM以亚纳米分辨率表征视紫红质蛋白质的结构和功能，视紫红质是视觉通路的G蛋白偶联受体（GPCR），在天然视杆外节膜和重构脂质膜中。 我们正在引入新的AFM模式，包括一个新的拉曼-AFM光谱仪器，以进一步揭示视紫红质分子组织，并探讨视紫红质信号和脂质膜环境之间的连接。 我们正在优化拉曼原子力显微镜检测灵敏度对单双层纳米结构域表征的蛋白质和脂质，使用共振和金属表面增强，新型的原子力显微镜的提示，和测量方式。 我们的目标是在纳米尺度上实现GPCR功能和膜结构之间更好的相关性。 我们还在与活细胞、细菌生物膜、仿生材料和基础表面科学相关的某些领域进行其他AFM相关研究。

Biological membranes, proteins, subcellular organelles, and other samples are being investigated via atomic force microscopy (AFM), Raman and fluorescent spectroscopy, and related biophysical approaches in this project covering several research collaborations. With further expansion of our technology, instrumentation, and data analysis methods, we have advanced a number of these applications in collaborations with many NIH intramural and extramural researchers. Major collaborations include: (1) We conpleted work on the effect of a mycobacterium tuberculosis-derived lipid, Lipoarabinomannan (LAM), on biological membranes with Drs. E. Hayakawa, F. Tokumasu, J.A. Dvorak (NIAID/NIH), and coworkers. Lipoarabinomannan is an integral component of the cell wall of the acid-fast bacillus mycobacterium tuberculosis (MTB). One hallmark of a tuberculosis infection is the ability of the bacterium to subvert the normal macrophage defense mechanism of the host immune response. LAM is reported to be involved in the inhibition of a phago-lysosomal fusion step of this defense mechanism. We have produced membranes in vitro composed of LAM and varying lipids to simulate phagosomal lipid membranes and quantified the effects of LAM using AFM and related studies. We found that LAM markedly alters membrane nanostructure and inhibits large unilamellar vesicle fusion, with possible implications for the survival of MTB and the fighting of uman tuberculosis disease. (2) We have investigated the macromolecular structure of a recombinant Plasmodium falciparum Merozoite Surface Protein 3 (MSP3) and more recently Plasmodium falciparum circumsporozoite protein (CSP) via AFM in collaboration with Dr. David Narum (NIAID, NIH) and coworkers. MSP3 and CSP are potential components of a human malaria vaccine. These protein antigens 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 conditions, finding mainly EcCSP monomers, but EcMSP3 monomers, dimers, and multimers with fluctuating folding of the secondary structural domains. Such structures should have implications for protein-protein interactions and human immunological response. (3) We have expanded our AFM studies of clathrin and clathrin coated vesicles (CCVs) in collaboration with Drs. Ralph Nossal (NICHD, NIH), 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. Here we have continued to develop and apply new schemes of atomic force microscopy (AFM) and related analyses toward the characterizations of triskelia and their assemblies. 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 dimers, are readily observed both dried on mica surface and showing shape flexibility 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 detected a considerable variability in triskelion conformation and clathrin cage nano-mechanics. 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. (4) We have continued our collaboration with Drs. Shui-Lin Niu, Drake Mitchell, Klaus Gawrisch (NIAAA, NIH) and colleagues, using AFM to characterize at sub-nanometer resolution the structure and function of the protein rhodopsin, a G-protein coupled receptor (GPCR) of the visual pathway, in native rod outer segment membranes and in reconstituted lipid membranes. We are introducing new AFM modalities, including a new Raman-AFM spectroscopy instrument, to further reveal rhodopsin molecule organization and to explore the connection between rhodopsin signaling and the lipid membrane environment. We are optimizing the Raman-AFM detecting sensitivity toward single bilayer nanometric domain characterization of proteins and lipids using resonance and metal surface enhancements, novel AFM tips, and measurement modality. We aim to achieve better correlations between GPCR function and membrane structure at nanometric scale. We are also conducting other AFM-related investigations in certain areas relating to live cells, bacterial biofilms, biomimicking materials, and fundamental surface sciences.