Using Homo-Polypeptides To Study Charge-Charge Interactions In Biomolecules

Charge-charge interactions in biomolecules are important for a range of biological processes such as enzyme-substrate and ligand-receptor recognition, auto-inhibition of signaling proteins, protein folding and balancing intrinsically disordered protein (IDP) function with aggregation. In order to more thoroughly understand the roles of charge-charge interactions in these systems, we use homo-polypeptides composed of glutamic acid and lysine residues. We use a range of calorimetric (ITC) and spectroscopic (FRET, CD, NMR) techniques to measure polypeptide binding and conformation under a range of solvent conditions such as pH, ionic strength and counter ion identity. In particular, using FRET to monitor such interactions allows us to achieve extremely high levels of sensitivity, but requires us to add an external fluorophores to the polypeptides. We describe the labeling and purification of polypeptides and determine the effect of the label on the interaction by comparing the thermodynamic properties of the labeled and unlabeled polypeptides. Results of this study show that the intermolecular interaction(s) through the charged side-chains of amino acids can significantly influence the backbone conformation of polypeptides. Our overall aim is to quantify the relative contributions of enthalpy and entropy in determining interaction mechanisms in highly charged regions of proteins.