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

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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.