Inverse methods for tuning dynamics of complex fluids

扩散探针KT尺度的特定蛋白质-蛋白质和蛋白质-碳水化合物相互作用这个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的蛋白质和碳水化合物之间的KT和纳米尺度的相互作用。自由扩散的胶体系综将被用作超灵敏探针，以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)。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、异质和带有共价连接和定向的蛋白质/碳水化合物的图案化底物附近的三维布朗漂移。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将通过利用扩散的胶体探针可获得的自然标尺来研究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)和分子长度尺度(Nm)(因此是弱力(~fn))。拟议研究的智力价值与将获得的对固定化蛋白质-蛋白质和蛋白质-碳水化合物相互作用的基本和技术见解有关。将被研究的具体生物分子相互作用包括：(1)钙依赖的同亲和异亲钙粘附素在支撑的脂类双层上的相互作用；(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相互作用受物理、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非常重要的。拟议工作的第一个任务是将蛋白质和碳水化合物共价结合/定向到硅胶和表面上，无论是否有支撑的脂双层。为了允许非特定和特定贡献的净电势的去卷积，第二个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)、竞争相互作用(例如抗体、单糖)和生化变化(例如不同类型、突变)如何影响共价连接/定向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，以直接和敏感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以及综合教育和宣传活动。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，为许多生物医学应用提供信息，包括诊断、设备、治疗、药物输送和组织工程。这样的理解为在生物医学应用中定量设计、控制和优化(正式设计)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/视频将纳入本科生热学和毕业胶体/聚合物选修课，执行拟议的研究将涉及对本科生和研究生的培训。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名学生的项目和公共博物馆演示。

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.