Loop flexibility is discussed as a factor that affects ligand binding affinity of SH3 domains. To test this hypothesis, we designed a mutant in which a proline in the RT-loop of the human Lck SH3-domain is replaced by glycine. The dynamics and ligand binding properties of wild-type and mutant LckSH3 were studied by fluorescence and NMR spectroscopy as well as molecular dynamics simulations. Although the mutated residue does not form direct contacts with the ligand, the mutation increases ligand affinity by a factor of eight. The mutant exhibits increased loop flexibility and enhanced sampling of binding-competent conformations. This effect is expected to facilitate ligand binding itself and might also allow formation of tighter contacts in the complex thus resulting in an increased binding affinity.