Biological Membranes and Atomic Force Microscopy

在这个项目中，正在通过原子力显微镜(AFM)和其他生物物理技术研究生物膜和相关系统，其中包括几个合作项目。(1)与NIAAA的科学家B.J.Litman博士和S.-L.Niu博士合作，我们的OD团队正在使用原子力显微镜(AFM)来表征视紫红质蛋白的结构和功能，该蛋白是一种G蛋白偶联受体(GPCR)，位于视觉通路的天然视杆外段膜中，以及在含有二棕榈酰磷脂酰胆碱(DPPC)膜的重组体系中。原子力显微镜成像揭示了亚纳米分辨率的视紫红质分子作为单体，并根据膜的物理状态和环境条件在不同的多聚体组织中。对于仿生膜，我们特别关注DDPC/DPPC/胆固醇三组分体系，其中DDPC(di-22：6n-3pc)是多不饱和的，由二十二碳六烯酸(DHA)衍生。AFM对微纳米结构的研究与差示扫描量热仪(DSC)的热力学信息相结合，探索视紫红质信号与脂膜环境之间的联系。(2)与NIAID科学家(J.A.Dvorak博士和F.Tokumasu博士)和一位校外研究员(康奈尔大学G.W.Feigenson教授)合作，我们完成了一系列关于脂膜的异质性的研究，包括1，2-二肉豆蔻-sn-甘油-3-磷胆碱(DMPC)、DPPC、二月桂酰磷脂酰胆碱(DLPC)和胆固醇(CHOL)。我们发现，通过大的单层囊泡(LUV)表面的吸附和融合，仿生膜很容易在原子扁平的云母载体上形成。然后，AFM成像显示了广泛的纳米膜域(参见。膜筏)的直径约为40纳米，这取决于胆固醇水平、DLPC和DPPC的比例以及其他相空间参数，例如云母基质的支撑强度。在这些研究中采用了数学分析，以全面了解细胞膜在其组成和天然相互作用的影响下的情况。与膜结构相联系，并与NIAID的科学家(J.A.Dvorak博士和T.Arie博士)合作，我们将对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，以表征恶性疟原虫感染和发育过程中的红细胞。我们发现，寄生虫感染显著改变了人体微循环中与疟疾发病机制潜在相关的细胞膜动力学。(3)与另一位NIAID科学家(J.Silver博士)合作，我们开发了生物膜的纳米能量学和结构描述，以了解融合孔动力学对许多细胞过程(如病毒入侵和囊泡运输)非常重要。(4)最后，与校外科学家合作(A.Sinz博士和O.Zschornig博士，大学)。在德国莱比锡，我们研究了一种融合多肽及其突变体的金属离子结合特性，这种结合蛋白是海胆受精过程中卵子-精子膜融合的关键。电喷雾电离傅里叶变换离子-回旋共振质谱仪(ESI-FTICRMS)测量了多肽/金属络合物，并结合数学模型对为什么Bindin/Cu2+抑制膜融合，而Bindin/Zn2+促进融合提供了更多的见解。

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.