Inverse methods for tuning dynamics of complex fluids

Truskett，托马斯M.CBET-1065357千吨级特异性蛋白质-蛋白质和蛋白质-碳水化合物相互作用的扩散探针本项目涉及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蛋白质和碳水化合物的千吨级和纳米级相互作用。自由扩散胶体的集合体将被用作超灵敏探针，以测量附着于胶体和平面表面的蛋白质/碳水化合物之间的平均力（PMF）的能量与分离依赖的蛋白质-蛋白质和蛋白质-碳水化合物势。通过使用倏逝波和视频显微镜，DCPM将监测蛋白质修饰的胶体的三维布朗漂移，这些胶体靠近具有共价连接和取向的蛋白质/碳水化合物的均匀、非均匀和图案化基底。因为扩散探针根据它们的相对能量采样空间位置，所以扩散探针的统计力学分析可以被解释为包括可重叠的非特异性（胶体、大分子）和特异性（残基、构象）贡献的净PMF。DCPM将通过利用扩散胶体探针可访问的自然测量仪来询问蛋白质和碳水化合物的相互作用，包括布朗时间尺度（a2 /D），热能（kT），分子长度尺度（nm）（因此弱力（~fN））。所提出的研究的智力价值与将获得固定化蛋白质-蛋白质和蛋白质-蛋白质的基础和技术见解有关。待研究的特异性生物分子相互作用包括：（1）钙离子依赖的亲钙和异钙钙粘附蛋白在支持的脂质双层上的相互作用，（2）在竞争性寡糖存在下的CD 44-透明质酸（HA）相互作用。钙粘蛋白是一种跨膜蛋白，其相互作用在决定细胞粘附的细胞过程中起关键作用，所述细胞过程包括例如组织形态发生、突触可塑性、细胞凋亡和癌症转移。CD 44是HA的主要细胞表面信号受体，HA是细胞外基质成分。因此，CD 44-HA相互作用调节细胞间粘附、细胞迁移、形态发生、细胞增殖、通过调节基因表达和RNA剪接的细胞信号传导、细胞分化和转移。最后，测量固定化的钙粘蛋白-钙粘蛋白和CD 44-HA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是重要的，所提出的工作的第一个任务是将蛋白质和碳水化合物共价连接/定向到二氧化硅胶体和表面上，无论是否有支持的脂质双层。为了对非特异性和特异性贡献的净电势进行去卷积，第二个任务是测量共价连接/取向的碳水化合物和蛋白质的相互作用如何受到不同溶液化学的影响（如离子、小分子）、物理构型（例如方向、空间组织）、竞争性互动（例如抗体、单糖）和生物化学变异（例如不同类型、突变）。最后一项任务是测量模型阵列上的非特异性和特异性相互作用，模型阵列由碳水化合物或共价连接/定向蛋白质的图案化区域组成。拟议目标的顺利完成将展示一种概念上的新方法，可以直接和灵敏地量化KT级非特异性和特异性相互作用，这些相互作用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。拟议研究的更广泛影响包括科技成果以及综合教育和推广活动。直接和灵敏地测量合成材料表面上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，包括诊断，设备，治疗，药物输送和组织工程。这种理解提供了一个基础，定量设计，控制和优化（正式工程师）的性质和行为的固定化蛋白质和碳水化合物在biomedicalapplications和提供新的见解超出了已知的试错发现。在教育方面，研究图像/视频将被纳入本科生热和研究生胶体/聚合物选修课程，拟议研究的执行将涉及培训本科生和研究生。在推广方面，涉及胶体光学显微镜的内容将被改编用于7-12名学生的课程和公共博物馆的演示。

Truskett, Thomas M.CBET-1065357Diffusing Probes of kT-scale Specific Protein-Protein and Protein-Carbohydrate InteractionsThis project is concerned with using Diffusing Colloidal Probe Microscopy (DCPM) to directlyand nonintrusively measure kT and nanometer scale interactions of surface immobilized proteins andcarbohydrates. Ensembles of freely diffusing colloids will be employed as ultra-sensitive probes tomeasure energy vs. separation dependent protein-protein and protein-carbohydrate potentials of meanforce (PMF) between proteins/carbohydrates attached to colloids and planar surfaces. By usingevanescent wave and video microscopy, DCPM will monitor three dimensional Brownian excursions ofprotein decorated colloids near homogeneous, heterogeneous, and patterned substrates with covalentlyattached and oriented proteins/carbohydrates. Because diffusing probes sample spatial positions accordingto their relative energies, statistical mechanical analyses of diffusing probes can be interpreted as netPMFs including superimposable non-specific (colloidal, macromolecular) and specific (residues,conformational) contributions. DCPM will interrogate protein and carbohydrate interactions by exploitingnatural gauges accessible with diffusing colloidal probes including Brownian time scales (a2 /D), thermalenergies (kT), and molecular length scales (nm) (and hence weak forces (~fN)).The intellectual merit of the proposed research is related to the fundamental and technologicalinsights that will be gained into immobilized protein-protein and protein-carbohydrate interactions.Specific biomolecular interactions to be investigated include: (1) Ca2+ dependent homophilic andheterophilic cadherin interactions on supported lipid bilayers, (2) CD44-hyaluronic acid (HA) interactionsin the presence of competing oligosaccharides. Cadherins are transmembrane proteins whose interactionsplay a critical role in determining cell adhesion in cellular processes including for example tissuemorphogenesis, synaptic plasticity, apoptosis, and cancer metastasis. CD44 is the main cell surfacesignaling receptor for HA, which is an extracellular matrix component. As a result, CD44-HA interactionsregulate cell-cell adhesion, cell migration, morphogenesis, cell proliferation, cell signaling via regulationof gene expression and RNA splicing, cell differentiation, and metastasis. Ultimately, measuring howimmobilized cadherin-cadherin and CD44-HA interactions are influenced by physical, chemical, andbiochemical 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 ontosilica colloids and surfaces either with or without supported lipid bilayers. To allow for deconvolution ofnon-specific and specific contributions to net potentials, the second task is to measure how theinteractions of covalently attached/oriented carbohydrates and proteins are influenced by varying solutionchemistries (e.g. ions, small molecules), physical configuration (e.g. orientation, spatial organization),competitive interactions (e.g. antibodies, monosaccharides), and biochemical variations (e.g. differenttypes, mutations). The last task is to measure non-specific and specific interactions on model arraysconsisting of patterned regions of carbohydrates or covalently attached/oriented proteins. Successfulcompletion of proposed objectives will demonstrate a conceptually new approach to directly andsensitively quantify kT-scale non-specific and specific interactions that control non-covalent, equilibriumbinding of proteins and carbohydrates immobilized on particles and surfaces.The broader impacts of the proposed research include scientific and technological outcomes aswell as integrated education and outreach activities. The ability to directly and sensitively measure weakinteractions between proteins and carbohydrate on synthetic material surfaces provides information toenable many biomedical applications involving diagnostics, devices, therapeutics, drug delivery, andtissue engineering. Such understanding provides a basis to quantitatively design, control, and optimize(formally engineer) the properties and behavior of immobilized proteins and carbohydrates in biomedicalapplications and provide new insights beyond what is known from trial-and-error discovery. In terms ofeducation, research images/videos will be incorporated into undergraduate thermo and graduatecolloid/polymer elective courses and execution of the proposed research will involve trainingundergraduate and graduate students. In terms of outreach, content involving optical microscopy ofcolloids will be adapted for use in programs for 7-12 students and public museum presentations.