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

ID：MPS/DMR/BMAT（7623）0907652 PI：贝克，大卫 ORG：田纳西州标题：多价碳水化合物-蛋白质结合表面与确定的密度和价模仿细胞膜该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。智力优点：糖萼的碳水化合物是第三个和最少研究的部分级联基因组学的蛋白质组学的糖组学，数字在生命系统中如此重要。糖萼是一层糖复合物，覆盖在真核细胞和原核细胞上，形成一个化学丰富的景观，在细胞类型之间变化。独特而复杂的形貌是由（a）寡糖结构的多样性与（B）分级组织的组合产生的，为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异性相互作用是必不可少的细胞粘附，运动，细胞？细胞通讯和识别环境中的化学成分。该项目的最初目的是开发一种模块化方法，用于多价碳水化合物配体的组合组装和表面展示，目的是生成与多聚体凝集素识别的层次模式相匹配的仿生配体呈现。拟议的化学将创建由三个不同模块组装的不同簇：功能化的碳水化合物配体连接到多价节点，这些节点反过来通过控制空间方向的灵活连接子来控制化合价。糖簇的最终组装将通过微流体试剂递送在纳米图案化表面上进行，从而允许糖簇阵列的直接组合合成。阵列开发后，该平台将用于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。最初，将测量荧光标记的凝集素对具有限定的化合价和间距的固定化碳水化合物的亲和力，指示针对每个特定受体的多价结合的最佳架构。研究还将采用基于AFM的方法来提供表面形貌和蛋白质识别的定量评估。最后，将开发一种基于FRET的检测方法，使用荧光标记的碳水化合物受体，以高分辨率检测碳水化合物聚集。更广泛的影响：所述项目将有益地影响多个层面的科学基础设施。 (1)在实验室中，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，以解决基本的科学问题。(2)为了进一步鼓励学生之间的跨学科学习，PI将与我们的同事合作，为多学科研究生课程提供讲座，吸引来自各个部门的学生，包括化学，物理，材料科学和化学工程（见附件）。(3)PI将通过在顶级期刊上的演讲和出版物向科学界和非专业人士传播这项研究。(4)最后，PI将把所描述的研究与几项旨在影响我们当地社区的外展计划相结合，如下所述。 主要活动将导致与UTK的学术推广和通信（OAOC）办公室正在进行的联合努力？美国文理学院。OAOC运营的外展计划包括有抱负的科学家参与研究和教育（ASPIRE），该计划将UTK研究人员派往社区学校进行互动，以及大学预科研究学者计划（PCRSP），当地高中生在UTK进行研究。 通过OAOC的努力，PI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%的学生被列为经济困难学生;（2）法拉格特高中（FHS），位于富裕的郊区，最近成立了一个科学学院，并翻新了实验室。在这笔赠款的支持下，PI将与BMS和FHS的科学教师一起扩大推广活动。目前和未来的活动包括：（1）作为PCRSP的一部分，高中学生在他们的实验室进行研究;（2）研究生研究人员将前往合作学校，以协助和加强实验室课程，作为ASPIRE计划的一部分;（三）PI将在当地学校举行的系列讲座中发表演讲，以提高人们对相关好处和令人兴奋的职业机会的认识进行科学研究。

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.