Characterizing Transient Protein-Protein Interactions by Trp-Cys Quenching and Computer Simulations

Transient protein-protein interactions occur frequently under the crowded conditions encountered in biological environments, yet they remain poorly understood. Here, tryptophan-cysteine quenching is introduced as an experimental approach that is ideally suited to characterize such interactions between proteins with minimal labeling due to its sensitivity to nano- to microsecond dynamics on sub-nanometer length scales. The experiments are paired with computational modeling at different resolutions including fully atomistic molecular dynamics simulations to provide interpretation of the experimental observables and add further insights at the molecular level. This approach is applied to model systems, villin variants and the drkN SH3 domain, in the presence of protein G crowders. It is demonstrated that Trp-Cys quenching experiments are able to not only distinguish between overall attractive and repulsive interactions between different proteins, but they can also discern variations in interaction preferences at different protein surface locations. The close integration between experiment and simulations also provides an opportunity to evaluate different molecular force fields for the simulation of concentrated protein solutions. Significance StatementBiological environments typically involve a variety of different proteins at very high concentrations where non-specific interactions are unavoidable. These interactions may go beyond simple crowding effects and involve transient contacts that may impact structure, dynamics, and ultimately function of proteins in vivo. While computer simulations have partially characterized such interactions, experimental data remain limited because established techniques are generally not well-suited to the characterization of dynamic processes on microsecond time and nanometer length scales. Tryptophan quenching by cysteine is introduced here as a new approach for studying transient protein encounters under concentrated conditions with the support of computational modeling. The study demonstrates that such experiments can resolve not just differences between different proteins but also residue-specific interaction preferences.