Competitively selected protein ligands pay their increase in specificity by a decrease in affinity.

Protein-ligand interactions characterise and govern the current state and fate of a living cell. The specificity of proteins is mainly determined by the relative affinities to each potential ligand. To investigate the consequences and potentials of ligands with increased specificity in comparison with ligands optimised solely for affinity, it was necessary to identify ligands that are optimised towards specificity instead of a barely optimised affinity to a given target. In the presented example, a modified phage display screening procedure yielded specific ligands for the LckSH3 domain. We found that increased specificity of one of the hereby obtained ligands for LckSH3 is achieved at the cost of a slightly reduced affinity to LckSH3 and a drastically reduced affinity to other SH3 domains. A surface plasmon resonance experiment simulating in vivo-like realistic competitive binding conditions exerted enhanced binding behaviour of the specific ligand under these binding conditions. The experimental data, together with a mathematical model describing the complex experimental situation, and theoretical considerations lead to the conclusion that increased specificity is achieved at the cost of reduced affinity, but after all, it pays if the ligand is applied under realistic, i.e. competitive, conditions.