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)我们继续与B.J.Litman博士和S.L.Niu博士(NIAAA，NIH)合作，使用原子力显微镜(AFM)在亚纳米分辨率下表征视紫红质蛋白质的结构和功能，视紫红质是一种G-蛋白质偶联受体(GPCR)，在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，di16：0pc)和多不饱和脂类如DDPC(di-22：6N-3pc)的重组膜中。我们在更广泛的自然视盘上获得了新的AFM数据，并在更广泛的条件下重建了视紫红质/膜系统。我们开发了新的AFM成像分析方法和生物物理模拟，以进一步揭示视紫红质分子的组织结构，并探索视紫红质信号与脂膜环境之间的联系。(2)我们与美国国立卫生研究院NICHD的Ralph Nossal博士以及德克萨斯大学圣安东尼奥分校的Eileen M.Lafer和K.Prasad博士合作，推进了我们对笼状蛋白涂层囊泡(CCV)的尖端AFM研究。CCV是重要的生物膜和多蛋白复合体，在受体介导的内吞作用和跨高尔基体网络的细胞内转运中发挥核心作用。我们用原子力显微镜和专门的生物物理数据分析方法测量了CCV的力学性能。我们已经确认了从电子显微镜中已知的拓扑型网状蛋白涂层结构，并进一步产生了CCV变形，作为对100pN范围内的压缩力的定量响应。将CCVS模拟为复合弹性薄壳，我们估计完整的CCVS的弯曲刚度约为其构成的外层笼状多面体晶格和内层磷脂膜的10倍。这表明，天然CCV中的接头蛋白层在其网状蛋白外壳与内脂类膜之间提供了部分偶联，内脂膜上负载了受体和配体。(3)利用原子力显微镜、生物物理数据分析、纳米技术等科学方法，继续我们的生物膜及相关研究。

Biological membranes and related systems are being investigated via atomic force microscopy (AFM) and other biophysical technologies in this project containing several collaborations. (1) We continue our collaboration with Drs. B. J. Litman and S.-L. Niu (NIAAA, NIH), using atomic force microscopy (AFM) to characterize at sub-nanometer resolutions 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 membranes with dipalmitoylphosphatidylcholine (DPPC, di16:0PC) and polyunsatuatrated lipids such as DDPC (di-22:6n-3PC). We have obtained new AFM data on a broader range of native vision disks and reconstituted rhodopsin/membrane system over a broader set of conditions. We have developed new AFM imaging analysis methods and biophysical simulations to further reveal rhodopsin molecule organization and to explore the connection between rhodopsin signaling and the lipid membrane environment. (2) We have advanced our cutting edge AFM studies of clathrin coated vesicles (CCVs) in collaboration with Dr. Ralph Nossal (NICHD, NIH) and Drs. Eileen M. Lafer and K. Prasad (University Texas, San Antonio). CCVs are important biological membrane and multi-protein complexes that play a central role in receptor-mediated endocytosis and intracellular trafficking from the trans-Golgi network. We have measured the mechanical properties of CCVs using AFM and dedicated biophysical data analysis methods. We have confirmed the topological clathrin coat structures known from electron microscopy and moved further to yield CCV deformation as a quantitative response to the compression force in the 100 pN range. Modeling CCVs as composite elastic thin shells, we estimate that the bending rigidity of intact CCVs is about 10 times that of its constituting outer clathrin polyhedral lattice and inner phospholipids membrane. This suggests that the adaptor protein layer in native CCVs provides a partial coupling between its clathrin coat and the inner lipid membrane loaded with receptors and ligands. (3) We continue our biological membrane and related studies using AFM, biophysical data analyses, nanotechnology, and other scientific methods.