Diffusing Probes of kT-scale Specific Protein-Protein & Protein-Carbohydrate Interactions

Bevan#1066254这个项目致力于使用扩散胶体探针显微镜(DCPM)直接和非侵入性地测量表面固定的蛋白质和碳水化合物之间的KT和纳米级相互作用。自由扩散的胶体系综将被用作超灵敏探针，用于测量附着在胶体和平面上的蛋白质/碳水化合物之间的能量与分离相关的蛋白质-蛋白质和蛋白质-碳水化合物之间的平均作用力(PMF)势。通过使用逝去波和视频显微镜，DCPM将监测蛋白质装饰胶体在均匀、异质和图案化的底物附近的三维布朗漂移，底物上有共价连接和定向的蛋白质/碳水化合物。由于扩散探针根据其相对能量对空间位置进行采样，扩散探针的统计力学分析可以解释为包括可叠加的非特定(胶体、大分子)和特定(残基、构象)贡献的净PMF。DCPM将利用扩散胶体探针可获得的自然尺度，包括布朗时间尺度(a2/D)、热能(KT)和分子长度尺度(Nm)(因此弱力(~fn))来研究蛋白质和碳水化合物的相互作用。拟议研究的智力价值与将从固定化蛋白质-蛋白质和蛋白质碳水化合物相互作用中获得的基本和技术见解有关。将被研究的特定生物分子相互作用包括：(1)依赖于钙离子的同亲和异亲钙粘附素在支撑的脂双层上的相互作用；(2)CD44-透明质酸(HA)在竞争寡糖存在下的相互作用。钙粘附素是一种跨膜蛋白，其相互作用在决定细胞黏附的细胞过程中起着关键作用，包括组织形态发生、突触可塑性、细胞凋亡和肿瘤转移。CD44是透明质酸的主要细胞表面信号受体，透明质酸是细胞外基质成分。因此，CD44-HA相互作用通过调节基因表达和RNA剪接、细胞分化和转移来调节细胞间的黏附、细胞迁移、形态发生、细胞增殖、细胞信号转导。最终，测量固定化钙粘蛋白-钙粘蛋白和CD44-HA的相互作用如何受到物理、化学和生化变量的影响对于理解基础生物学和生物医学应用非常重要。拟议工作的第一个任务是将蛋白质和碳水化合物共价连接/定向到硅胶和表面上，无论是否有支撑的脂双层。为了实现对净电势的非特定和特定贡献的去卷积，第二项任务是测量不同的溶液化学(例如离子、小分子)、物理构型(例如取向、空间组织)、竞争性相互作用(例如抗体、单糖)和生化变化(例如不同的类型、配方)如何影响共价连接/定向的碳水化合物和蛋白质的相互作用。最后一项任务是测量由碳水化合物或共价连接/定向蛋白质的图案化区域组成的模型阵列上的非特定和特定相互作用。拟议目标的成功完成将展示一种概念上的新方法，以直接和灵敏地量化控制固定在颗粒和表面上的蛋白质和碳水化合物的非共价、平衡结合的KT规模的非特定和特定相互作用。拟议研究的更广泛影响包括科技成果和综合教育。能够直接和灵敏地测量合成材料表面蛋白质和碳水化合物之间的弱相互作用的能力提供了信息，使许多生物医学应用成为可能，涉及诊断、设备、治疗、药物输送和组织工程。这样的理解为定量设计、控制和优化(正式设计)生物医学应用中固定化蛋白质和碳水化合物的性质和行为提供了基础，并提供了超越试错发现的新见解。在教育方面，研究图像/视频将被纳入本科生热学和研究生胶体/聚合物选修课，执行拟议的研究将涉及培训本科生和研究生。在外展方面，涉及胶体光学显微镜的内容将被用于7-12名学生的项目和公共博物馆演示。

Bevan#1066254This project is concerned with using Diffusing Colloidal Probe Microscopy (DCPM) to directly and nonintrusively measure kT and nanometer scale interactions of surface immobilized proteins and carbohydrates. Ensembles of freely diffusing colloids will be employed as ultra-sensitive probes to measure energy vs. separation dependent protein-protein and protein-carbohydrate potentials of mean force (PMF) between proteins/carbohydrates attached to colloids and planar surfaces. By using evanescent wave and video microscopy, DCPM will monitor three dimensional Brownian excursions of protein decorated colloids near homogeneous, heterogeneous, and patterned substrates with covalently attached and oriented protein/carbohydrates. Because diffusing probes sample spatial positions according to their relative energies, statistical mechanical analyses of diffusing probes can be interpreted as net PMFs including superimposable non-specific (colloidal, macromolecular) and specific (residues, conformational) contributions. DCPM will interrogate protein and carbohydrate interactions by exploiting natural gauges accessible with diffusing colloidal probes including Brownian time scales (a2/D), thermal energies (kT), and molecular length scales (nm) (and hence weak forces (~fN)).The intellectual merit of the proposed research is related to the fundamental and technological insights that will be gained into immobilized protein-protein and protein carbohydrate interactions. Specific biomolecular interactions to be investigated include: (1) Ca2+ dependent homophilic and heterophilic cadherin interactions on supported lipid bilayers, (2) CD44-hyaluronic acid (HA) interactions in the presence of competing oligosaccharides. Cadherins are transmembrane proteins whose interactions play a critical role in determining cell adhesion in cellular processes including for example tissue morphogenesis, synaptic plasticity, apoptosis, and cancer metastasis. CD44 is the main cell surface signaling receptor for HA, which is an extracellular matrix component. As a result, CD44-HA interactions regulate cell-cell adhesion, cell migration, morphogenesis, cell proliferation, cell signaling via regulation of gene expression and RNA splicing, cell differentiation, and metastasis. Ultimately, measuring how immobilized cadherin-cadherin and CD44-HA interactions are influenced by physical, chemical, and biochemical variables is important to understanding fundamental biology and biomedical applications.The first task of the proposed work is to covalently attach/orient proteins and carbohydrates onto silica colloids and surfaces either with or without supported lipid bilayers. To allow for deconvolution of non-specific and specific contributions to net potentials, the second task is to measure how the interactions of covalently attached/oriented carbohydrates and proteins are influenced by varying solution chemistries (e.g. ions, small molecules), physical configuration (e.g. orientation, spatial organization), competative interactions e.g. antibodies, monosaccharides), and biochemical variations (e.g. different types, multations). The last task is to measure non-specific and specific interactions on model arrays consisting of patterned regions of carbohydrates or covalently attached/oriented proteins. Successful completion of proposed objectives will demonstrate a conceptually new approach to directly and sensitively quantify kT-scale non-specific and specific interactions that control non-covalent, equilibrium binding of proteins and carbohydrates immobilized on particles and surfaces.The broader impacts of the proposed research include scientific and technological outcomes as well as integrated education. The ability to directly and sensitively measure weak interactions between proteins and carbohydrate on synthetic material surfaces provides information to enable many biomedical applications involving diagnostics, devices, therapeutics, drug delivery, and tissue engineering. Such understanding provides a basis to quantitatively design, control, and optimize (formally engineer) the properties and behavior of immobilized proteins and carbohydrates in biomedical applications and provide new insights beyond what is known from trial-and-error discovery. In terms of education, research images/videos will be incorporated into undergraduate thermo and graduate colloid/polymer elective courses and execution of the proposed research will involve training undergraduate and graduate students. In terms of outreach, content involving optical microscopy of colloids will be adopted for use in programs for 7-12 students and public museum presentations.