Protein-Ligand Association

Fluorescence measurements are carried out in 50 mM Hepes/KOH (pH 7.2), 100 mMKCl, 0.5 mMEDTA, and 2 mMDTT at 20°. To 0.1 or 0.05 /iM mouse eIF4E(28-217) (i.e., the first 27 amino acid residues are truncated) (Niedzwiecka et al., 2002) are added solutions of increasing cap analog concentrations. For this protein—ligand association, an excitation wavelength of 280 nm and an emission wavelength of 337 nm are used. 

W discuss three examples of "drug target" interactions (l)biotin-avidin (2) dihydrofolate reductase-trimethoprim, and (3)DNA-in-tercalator. The first is the strongest characterized protein-ligand association, the second a prototype enzyme-inhibitor interaction, and the third describes drugs interacting with nucleic acids. 

The unequivocal demonstration of the importance of dispersion forces in protein-ligand association is difficult, since individual atom-atom energies are rather small and it is the sum of many atom-atom interactions which determines whether an association is given by the dispersion interactions or not. 

Held, M., Noe, F. (2012) Calculating kinetics and pathways of protein-ligand association. EurJ Cell Biol, 91 (4), 357-364. 

Surfece plasmon resonance (SPR) measures adsorption of material onto a planar metal (typically gold) surface and provides a very powerful tool for protein binding assays by analyzing the protein-ligand association and dissociation. Typically, pure protein is immobilized in buffer solution at different densities (according to the chips) using standard coupling reactions followed by ethanol-amine deactivation of the surfaces. A control surface is also prepared in the same manner in the absence of protein. 

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