Multivalent Carbohydrate-Protein Binding on Surfaces with Defined Density and Valency that Mimics Cellular Membranes

ID：MPS/DMR/BMAT(7623)0907652 PI：BAKER，David ORG：Tennessee标题：多价碳水化合物-蛋白质结合在具有限定密度和价态的表面上，模仿细胞膜该奖项由2009年美国复苏和再投资法案(公共法律第111-5号)资助。糖萼是覆盖在真核和原核细胞上的一层糖共轭化合物，形成了一种丰富的化学景观，不同的细胞类型不同。独特、复杂的形貌是由(A)寡糖结构的多样性和(B)层级组织的组合产生的，为被称为凝集素的多聚体碳水化合物结合蛋白对细胞表面的高度特异性识别提供了基础。通过细胞-表面碳水化合物的特定相互作用对于细胞黏附、运动、细胞-细胞交流和识别环境中的化学成分是必不可少的。该项目的最初目标是开发一种用于多价碳水化合物配体的组合组装和表面展示的模块化方法，目的是生成与多聚体凝集素识别的层级模式相匹配的仿生配体展示。拟议的化学将创建由三个不同模块组装的不同簇：连接到多价节点的功能化碳水化合物配体，这些多价节点反过来通过控制空间取向的灵活连接物来管理价态。糖簇的最终组装将通过微流控试剂输送在纳米颗粒表面进行，从而允许直接、组合地合成糖簇阵列。随着阵列的发展，这个平台将被用来定量碳水化合物受体的结合，以阐明多价细胞-表面相互作用。首先，将测量荧光标记的凝集素与具有限定价态和间距的固定化碳水化合物的亲和力，表明每个特定受体的多价结合的最佳结构。研究还将使用基于AFM的方法来提供表面形貌的定量评估和蛋白质的识别。最后，将开发一种基于荧光标记碳水化合物受体的FRET分析，以高分辨率检测碳水化合物聚集。BROADER影响：所述项目将在多个层面上有益于影响科学基础设施。(1)在实验室，研究生、博士后和本科生将受益于这项研究的跨学科培训，该研究结合了合成化学、生物化学、材料科学和纳米技术以及分析技术来解决基本的科学问题。(2)为进一步鼓励学生进行跨学科学习，督导主任会与我们的同事合作，为一个跨学科的研究生课程提供讲座，吸引来自不同系的学生，包括化学、物理、材料科学和化学工程(见附信)。(3)PIS将通过在一流期刊上发表演讲和发表文章，向科学界和非科学界传播这项研究。(4)最后，督导人员将把上述研究与数项外展计划结合起来，以期影响我们的本地社区，如下所述。主要活动将由与加州大学S文理学院学术推广与交流办公室(OAOC)持续共同努力开展。OAOC实施的外展计划包括有抱负的科学家参与研究和教育计划(ASPIRE)，该计划派遣UTK研究人员与社区学校互动，以及大学预科研究学者计划(PCRSP)，当地高中生在UTK进行研究。在OAOC的推动下，PIS与当地两所学校的科学教师建立了关系：(1)比尔登中学(BMS)，44%的学生被归类为经济困难学生；(2)法拉格特高中(FHS)，位于富裕的郊区，最近成立了一所科学学院，并翻新了实验室。在这笔赠款的支持下，私立学校将扩大与波士顿大学和FHS的科学教师共同设计的外展活动。目前和未来的活动包括：(1)作为PCRSP的一部分，接待高中生在其实验室开展研究；(2)研究生研究人员将前往合作学校，协助和加强实验室课程，作为ASPIRE计划的一部分；(3)PIs将在当地学校举办的系列讲座上发表演讲，以提高对与科学研究相关的好处和令人兴奋的职业机会的认识。

ID: MPS/DMR/BMAT(7623) 0907652 PI: Baker, David ORG: TennesseeTitle: Multivalent Carbohydrate-Protein Binding on Surfaces with Defined Density and Valency that Mimics Cellular MembranesThis award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).INTELLECTUAL MERIT: Carbohydrates of the glycocalyx are the third and least studied part of the cascade of genomics to proteomics to glycomics that figure so importantly in living systems. The glycocalyx is a layer of glycoconjugates that coats both eukaryotic and prokaryotic cells, forming a chemically rich landscape that varies among cell types. Distinctive, complex topographies are generated from a combination of (a) diversity in oligosaccharide structures with (b) hierarchical organization, providing the basis for highly specific recognition of cell surfaces by multimeric carbohydrate-binding proteins called lectins. Specific interaction through cell-surface carbohydrates is essential for cell adhesion, motility, cell?cell communication and recognition of chemical components in the environment. The initial aim of the project is to develop a modular approach for the combinatorial assembly and surface display of multivalent carbohydrate ligands, with the aim of generating biomimetic ligand presentations that match the hierarchical patterns recognized by multimeric lectins. The proposed chemistry will create diverse clusters assembled from three distinct modules: functionalized carbohydrate ligands joined to multivalent nodes that, in turn, govern valency through flexible linkers that control spatial orientation. Final assembly of the glycoclusters will be performed on nanopatterned surfaces through microfluidic reagent delivery, allowing for the direct, combinatorial synthesis of glycocluster arrays. Following array development, this platform will be employed to quantitate the binding of carbohydrate receptors in order to elucidate multivalent cell-surface interactions. Initially, the affinities of fluorescent-tagged lectins for immobilized carbohydrates with defined valency and spacing will be measured, indicating the optimal architecture for multivalent binding for each particular receptor. Studies will also employ AFM-based methods to provide quantitative assessment of surface topography and recognition by proteins. Finally, a FRET-based assay using fluorescent-tagged carbohydrate receptors will be developed to detect carbohydrate clustering with high resolution.BROADER IMPACTS: The project as described will beneficially impact scientific infrastructure at multiple levels. (1) In the laboratory, graduate, post-doctoral and undergraduate students will benefit from the interdisciplinary training in this research, which combines synthetic chemistry, biochemistry, materials science and nanotechnology, and analytical techniques to address fundamental scientific questions. (2) To further encourage interdisciplinary learning among our students, the PIs will work with our colleagues by contributing lectures for a multidisciplinary graduate-level course that attracts students from various departments, including Chemistry, Physics, Materials Science, and Chemical Engineering (see attached letter). (3) The PIs will disseminate this research to the scientific and lay communities via presentations and publications in top-notch journals. (4) Finally, the PIs will integrate the described research with several outreach initiatives aimed at impacting our local community as described below. The primary activities will result from an ongoing joint effort with the Office of Academic Outreach and Communications (OAOC) of UTK?s College of Arts and Sciences. The OAOC operates outreach programs including Aspiring Scientists Participating In Research and Education (ASPIRE), which sends UTK researchers out to interact with community schools, and the Pre-Collegiate Research Scholars Program (PCRSP), in which local high school students conduct research at UTK. Through efforts facilitated by the OAOC, the PIs have formed relationships with science teachers at two local schools: (1) Bearden Middle School (BMS), at which 44% of students are classified as economically disadvantaged; and (2) Farragut High School (FHS), which is located in an affluent suburban area and recently launched a science academy and renovated its laboratories. With support from this grant, the PIs will extend outreach activities devised in conjunction with science teachers from BMS and FHS. Current and future activities include: (1) hosting of high school students to conduct research in their labs as part of the PCRSP; (2) graduate student researchers will travel to partner schools to assist with and enhance laboratory courses as part of the ASPIRE program; (3) the PIs will give presentations at lecture series held at local schools to raise awareness about the benefits and exciting career opportunities associated with scientific research.