Inverse methods for tuning dynamics of complex fluids

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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.