Photocontrolling Protein-Peptide Interactions: From Minimal Perturbation to Complete Unbinding.

An azobenzene-derived photoswitch has been covalently cross-linked to two sites of the S-peptide in the RNase S complex in a manner that the α-helical content of the S-peptide reduces upon cis-to-trans isomerization of the photoswitch. Three complementary experimental techniques have been employed, isothermal titration calorimetry, circular dichroism spectroscopy and intrinsic tyrosine fluorescence quenching, to determine the binding affinity of the S-peptide to the S-protein in the two states of the photoswitch. Five mutants with the photoswitch attached to different sites of the S-peptide have been explored, with the goal to maximize the change in binding affinity upon photoswitching, and to identify the mechanisms that determine the binding affinity. With regard to the first goal, one mutant has been identified, which binds with reasonable affinity in the one state of the photoswitch, while specific binding is completely switched off in the other state. With regard to the second goal, accompanying molecular dynamics simulations combined with a quantitative structure activity relationship revealed that the α-helicity of the S-peptide in the binding pocket correlates surprisingly well with measured dissociation constants. Moreover, the simulations show that both configurations of all S-peptides exhibit quite well-defined structures, even in apparently disordered states.