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Intact Protein and Biomolecular Interactions

In many cases, metabolites function as important effector molecules that impact biological activity (e.g., gene transcription, enzyme inhibition) as reflected by endogenous cellular metabolites, as well as numerous extrinsic compounds, including prescription drugs, dietary phytochemicals, microflora metabolites and environmental toxins. Our group is interested in developing new assays for studying biomolecular interactions between protein and small molecules when using CE as a way to assess binding affinity, conformational stability and mechanism(s) of action. These studies are relevant for high-quality drug screening due to the high attrition rate of conventional strategies for drug development. Also, intact protein analysis using CE-MS represents a new research initiative in our research group for better characterization of protein heterogeneity and post-translational modifications (e.g., glycation, phosphorylation, methylation, carboxylation), including medical foods and biologics for treatment of human diseases.

Figure 1-Biomolecular Interactions

Fig. 1. A label-free assay for determination of the thermodynamic stability of a cAMP-binding protein in free solution based on dynamic ligand stripping and protein denaturation in urea during electormigration when using CE with direct UV detection.

Figure 2-Biomolecular Interactions

Fig. 2. A label-free assay for direct determination of the enzymatic activity and coupling efficiency of CYP 450 isoforms for various substrates when using CE with direct UV detection with a short-end capillary outlet injection under reversed polarity. 

 

Recommended References:

  1. M. Shanmuganathan* and P. Britz-McKibbin “Functional Screening of Pharmaceutical Chaperones via Restoration of Enzyme Activity upon Denaturation” Biochemistry. 2012, 51:7651-7653.
  2. J. Harskamp,* P. Britz-McKibbin and J. Y. Wilson “Functional Screening of Cytochrome P450 Activity and Uncoupling by Capillary Electrophoresis” Anal. Chem. 2012, 84:862-866.
  3. M. Shanmugananthan* and P. Britz-McKibbin “Inhibitor Screening of Pharmacological Chaperones for Lysosomal b-Glucocerebrosidase by Capillary Electrophoresis” Anal. Bioanal. Chem. 2011, 399, 2843-2853.
  4. J.M.A. Gavina,* M.T. Mazhab-Jafari,* G. Melacini and P. Britz-McKibbin “Label-free Assay for Thermodynamic Analysis of Protein-ligand Interactions: A Multivariate Strategy for Allosteric Ligand Screening” Biochemistry. 2009, 48, 223-225.