This project is concerned with the application of a conceptually new method, 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 potentials of mean force (PMF) between proteins/carbohydrates attached to colloids and planar surfaces. By combining evanescent wave and video microscopy, DCPM will monitor three dimensional Brownian excursions of protein decorated colloids near homogeneous, heterogeneous, and patterned substrates also with covalently attached and oriented proteins/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, geometry) contributions. Consistent with the nanotechnology paradigm, DCPM will interrogate protein and carbohydrate interactions by exploiting natural gauges for time (a2/D), energy (kT), force (fN), and length (nm) associated with diffusing colloidal probes.
The intellectual merit of the research is broadly related to the fundamental insights that will be gained into immobilized protein-protein and protein-carbohydrate interactions (i.e. PMFs) with practical relevance to nanobiotechnologies (e.g. chemical sensors, protein arrays). Several biomolecular interactions will be investigated including: (1) maltose binding protein (MBP) with transmembrane aspartate receptor (Tar), (2) MBP with amylose, and (3) ConA with amylose. The first two interactions mediate E. coli maltose chemotaxis, which is one of the most thoroughly studied experimental models for chemical recognition/response by organisms with broad relevance to problems ranging from drug interactions to chemical detection. The last interaction will be studied partly because it is an inexpensive, commercially available, and well characterized model system that can be used to optimize initial DCPM experiments, but also because of its practical relevance to glucose sensing (i.e. diabetes control) and its complementarity to the second interaction. The first task of the proposed work is to covalently attach/orient proteins and carbohydrates onto gold (Au) colloids and either Au or silica surfaces (purified and characterized proteins provided by the collaborator). Building on preliminary DCPM measurements of non-specific macromolecular interactions, the second task is to measure interactions of covalently attached/oriented carbohydrates and proteins with various mutations and conformations to allow for deconvolution of non-specific and specific contributions to net potentials. The last task is to measure non-specific and specific interactions on a model array consisting of patterned regions of carbohydrates or covalently attached/oriented proteins. Successful completion of proposed objectives will demonstrate a new technology (DCPM) to directly and sensitively quantify weak 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 are related to the use of visual research content to provide intuitive educational experiences for students at all levels and to allow for authentic and guided inquiry experiences in the classroom. Educational tasks will involve integrating visual content (e.g. images, videos, renderings) generated in the proposed research in courses and outreach activities. In terms of classroom teaching, research images and videos will be incorporated into undergraduate and graduate colloid/polymer elective courses and an undergraduate thermodynamics core course. In terms of outreach, content involving optical microscopy of colloids will be adapted for use in programs for 7-12 grade teachers and students and for integration into web based modules used in core chemical engineering courses.
