Diffusing Probes of kT-scale Specific Protein-Protein & Protein-Carbohydrate Interactions

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Bevan#1066254This project is concerned with using Diffusing Colloidal Probe Microscopy (DCPM) to directly and nonintrusively measure kT and nanometer scale interactions of surface immobilized proteins and carbohydrates. Ensembles of freely diffusing colloids will be employed as ultra-sensitive probes to measure energy vs. separation dependent protein-protein and protein-carbohydrate potentials of mean force (PMF) between proteins/carbohydrates attached to colloids and planar surfaces. By using evanescent wave and video microscopy, DCPM will monitor three dimensional Brownian excursions of protein decorated colloids near homogeneous, heterogeneous, and patterned substrates with covalently attached and oriented protein/carbohydrates. Because diffusing probes sample spatial positions according to their relative energies, statistical mechanical analyses of diffusing probes can be interpreted as net PMFs including superimposable non-specific (colloidal, macromolecular) and specific (residues, conformational) contributions. DCPM will interrogate protein and carbohydrate interactions by exploiting natural gauges accessible with diffusing colloidal probes including Brownian time scales (a2/D), thermal energies (kT), and molecular length scales (nm) (and hence weak forces (~fN)).The intellectual merit of the proposed research is related to the fundamental and technological insights that will be gained into immobilized protein-protein and protein carbohydrate interactions. Specific biomolecular interactions to be investigated include: (1) Ca2+ dependent homophilic and heterophilic cadherin interactions on supported lipid bilayers, (2) CD44-hyaluronic acid (HA) interactions in the presence of competing oligosaccharides. Cadherins are transmembrane proteins whose interactions play a critical role in determining cell adhesion in cellular processes including for example tissue morphogenesis, synaptic plasticity, apoptosis, and cancer metastasis. CD44 is the main cell surface signaling receptor for HA, which is an extracellular matrix component. As a result, CD44-HA interactions regulate cell-cell adhesion, cell migration, morphogenesis, cell proliferation, cell signaling via regulation of gene expression and RNA splicing, cell differentiation, and metastasis. Ultimately, measuring how immobilized cadherin-cadherin and CD44-HA interactions are influenced by physical, chemical, and biochemical 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 onto silica colloids and surfaces either with or without supported lipid bilayers. To allow for deconvolution of non-specific and specific contributions to net potentials, the second task is to measure how the interactions of covalently attached/oriented carbohydrates and proteins are influenced by varying solution chemistries (e.g. ions, small molecules), physical configuration (e.g. orientation, spatial organization), competative interactions e.g. antibodies, monosaccharides), and biochemical variations (e.g. different types, multations). The last task is to measure non-specific and specific interactions on model arrays consisting of patterned regions of carbohydrates or covalently attached/oriented proteins. Successful completion of proposed objectives will demonstrate a conceptually new approach to directly and sensitively quantify kT-scale non-specific and specific interactions that control non-covalent, equilibrium binding of proteins and carbohydrates immobilized on particles and surfaces.The broader impacts of the proposed research include scientific and technological outcomes as well as integrated education. The ability to directly and sensitively measure weak interactions between proteins and carbohydrate on synthetic material surfaces provides information to enable many biomedical applications involving diagnostics, devices, therapeutics, drug delivery, and tissue engineering. Such understanding provides a basis to quantitatively design, control, and optimize (formally engineer) the properties and behavior of immobilized proteins and carbohydrates in biomedical applications and provide new insights beyond what is known from trial-and-error discovery. In terms of education, research images/videos will be incorporated into undergraduate thermo and graduate colloid/polymer elective courses and execution of the proposed research will involve training undergraduate and graduate students. In terms of outreach, content involving optical microscopy of colloids will be adopted for use in programs for 7-12 students and public museum presentations.