Development and application of analytical methodologies to study carbohydrate-modifying and carbohydrate-binding proteins

This work describes the development and application of analytical techniques to study protein function and characterize interactions in protein-ligand complexes. The first part of this work describes the development of small-scale enzyme assays. Using a novel nanopipettor, which was shown capable of tunable and reproducible nanolitre sampling, combined with cell manipulation and the ultrasensitivity of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF), a single cell enzyme assay was developed. Individual Sf9 cells were assayed for the enzyme alpha-glucosidase II and were found to be very heterogeneous with respect to their levels of alpha-glucosidase II activity. The most important benefit afforded by this technique is that a single cell lysate can be repeatedly sampled, which was demonstrated in the time-course study of alpha-glucosidase II activity in a single Sf9 cell.; Nanoflow electrospray (nanoES), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) was used to determine the association thermochemistry of two protein-ligand systems. The ligand specificity of the monoclonal antibody (mAb) CS-35 towards a polysaccharide cell wall component in Mycobacterium tuberculosis was investigated. Determination of the association thermochemistry for a series of ligands allowed the key residues in the interaction to be identified. A nanoES-MS study was also performed on the blood group enzyme, GTB, representing the first direct thermodynamic study of this enzyme. It was shown that GTB exists exclusively as a dimer in aqueous solutions (pH 7) with two equivalent acceptor binding sites. A study of GTB2 interacting with donor and donor fragments, both alone and in the presence of a divalent metal cofactor, concluded that GTB 2 possesses a weak binding affinity for its native donor in the absence of cofactor. The metal cofactor also exhibited a drastic effect on catalysis and it was suggested that the biologically relevant cofactor is Mn +2. Using a novel gas-phase assay, based on time-resolved tandem MS, the two GTB2 acceptor sites were found to maintain their equivalency in the gas-phase. The gas-phase assay was used to confirm that the binding site equivalency in the homotetramer of streptavidin was also maintained upon transfer to the gas-phase. This suggests that time-resolved tandem MS can be used to evaluate binding-site equivalency for protein-ligand complexes in solution.