Biological Membranes and Atomic Force Microscopy

生物膜和相关系统正在研究通过原子力显微镜（AFM）和其他生物物理技术在这个项目包含几个合作。(1)与NIAAA科学家（B博士）合作。J. Litman和S.- L. Niu），我们的OD团队正在使用原子力显微镜（AFM）来表征视紫红质（一种G蛋白偶联受体（GPCR））在视觉通路的天然视杆细胞外段膜中以及在具有二棕榈酰磷脂酰胆碱（DPPC，di 16：0 PC）膜的重建系统中的结构和功能。原子力显微镜成像揭示了单个视紫红质分子在亚纳米分辨率作为单体和各种多聚体组织取决于膜的物理状态和环境条件。对于仿生膜，我们特别关注三组分DDPC/DPPC/胆固醇系统，其中DDPC（di-22：6 n-3 PC）是多不饱和的，并且衍生自二十二碳六烯酸（DHA）。原子力显微镜的研究微米和纳米结构正在结合从差示扫描量热法（DSC）的热力学信息，探索视紫红质信号和脂质膜环境之间的连接。(2)与NIAID科学家（Drs. J.A. Dvorak和F. Tokumasu）和一名校外调查员（G. W. Feigenson，Cornell Univ.），我们已经总结了一系列关于脂质膜的异质性的研究，包括1，2-二肉豆蔻酰-sn-甘油基-3-磷酸胆碱（DMPC）、DPPC、二月桂酰磷脂酰胆碱（DLPC）和胆固醇（chol）。我们发现，仿生膜容易形成原子级平坦的云母支持通过大单层囊泡（LUV）表面吸附和融合。然后AFM成像揭示了广泛的纳米级膜结构域（c.f.膜筏），直径约为40纳米，取决于胆固醇水平、DLPC和DPPC比率以及其他相空间参数，如云母基底支撑的强度。在这些研究中采用数学分析，以全面了解其组成和天然相互作用的影响下的细胞膜。与膜结构有关，并与NIAID科学家（Drs. J.A. Dvorak和T. Arie），我们将闪烁和边缘抖动现象的视频显微镜图像分析扩展到整个细胞水平，以表征恶性疟原虫疟疾感染和发育期间的红细胞。我们发现，寄生虫感染显着改变细胞膜动力学在潜在的相关性疟疾疾病的机制，在人类微循环。(3)与另一位NIAID科学家（J.银博士）合作，我们开发了生物膜的纳米级能量和结构描述，以了解对许多细胞过程（如病毒入侵和囊泡运输）重要的融合孔动力学。(4)最后，与校外科学家（A。Sinz和O. Zschornig，Univ. Leipzig，德国），我们研究了融合肽及其突变体的金属离子结合特性，融合肽及其突变体来源于蛋白质结合蛋白，这对海胆受精过程中卵-精膜融合至关重要。肽/金属复合物的电喷雾电离傅里叶变换离子回旋共振质谱（ESI-FTICRMS）测量与数学建模一起增加了关于为什么bindin/Cu 2+抑制膜融合，但bindin/Zn 2+增强融合的见解。

Biological membranes and related systems are being investigated via atomic force microscopy (AFM) and other biophysical technologies in this project containing several collaborations. (1) Collaborating with NIAAA scientists (Drs. B. J. Litman and S.-L. Niu), our OD team is using atomic force microscopy (AFM) to characterize structure and function of the protein rhodopsin, a G-protein coupled receptor (GPCR), in native rod outer segment membranes of the vision pathway, and in reconstituted systems with dipalmitoylphosphatidylcholine (DPPC, di16:0PC) membranes. The AFM imaging has revealed individual rhodopsin molecules at sub-nanometer resolutions as monomers and in various multimeric organizations depending upon the physical state of the membranes and environmental conditions. For biomimicking membranes, we are paying particular attention to the three-component DDPC/DPPC/cholesterol system, where DDPC (di-22:6n-3PC) is polyunsatuatrated and derived from docosahexaenoic acid (DHA). AFM studies of micro- and nano-structures are being combined with thermodynamic information from differential scanning calorimetry (DSC) to explore the connection between rhodopsin signaling and the lipid membrane environment. (2) Collaborating with NIAID scientists (Drs. J.A. Dvorak and F. Tokumasu) and an extramural investigator (Prof. G. W. Feigenson, Cornell Univ.), we have concluded a set of studies on heterogeneities of lipid membranes involving 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), DPPC, dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). We found that biomimicking membranes readily form on atomically flat mica support via large unilamellar vesicle (LUV) surface adsorption and fusion. AFM imaging then revealed extensive nanoscopic membrane domains (c.f. membrane rafts) with diameters around 40 nanometers depending on cholesterol level, DLPC and DPPC ratio, and other phase space parameters such as the strength of mica substrate support. Mathematical analyses were employed in these studies toward a comprehensive understanding of the cellular membrane under influence of its composition and native interactions. Linked to membrane structures and also in collaboration with NIAID scientists (Drs. J.A. Dvorak and T. Arie), we extended our video microscope image analyses of flicker and edge dithering phenomena to the whole cell level to characterize red blood cells during the Plasmodium falciparum malaria infection and development. We found that the parasitic infection markedly modifies cell membrane dynamics in potential relevance to malaria disease mechanism in human microcirculations. (3) Collaborating with another NIAID scientist (Dr. J. Silver), we have developed nanoscopic energetic and structural descriptions of biological membranes to understand fusion pore dynamics important to many cellular processes such as virus invasion and vesicular trafficking. (4) Finally, collaborating with extramural scientists (Drs. A. Sinz and O. Zschornig, Univ. Leipzig, Germany), we have investigated the metal ion binding properties of a fusogenic peptide and its mutants derived from the protein bindin, which is crucial for egg-sperm membrane fusion during sea urchin fertilization. Electrospray Ionization Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry (ESI-FTICRMS) measurements of peptide/metal complexes together with mathematical modeling have added insights on why bindin/Cu2+ suppresses membrane fusion, but bindin/Zn2+ enhances fusion.