Tuning of Structure-Function Relationships by Macromolecular Crowding

The cellular interior is crowded with high concentrations of macromolecules and small organic solutes and strict maintenance of this environment, where ∼30% of the total volume is occupied, is essential to cellular function. The volume occupied by these molecules confines the 3D space in which any particular protein can reside and is thought to modulate protein-protein interactions by affecting diffusional encounter and reaction rates. In this work, we investigate the mechanistic consequences of crowded environments on the structure-function relationship of the ubiquitous protein calmodulin (CaM). CaM is highly flexible and dynamic, making it particularly susceptible to crowded environments, and its conformational plasticity is essential for accommodating binding to its 300 identified targets. We determined that crowded environments, created with purified polymer systems and sucrose, stabilize compact conformers of CaM, reduce translational and rotational diffusion, modulate association and dissociation kinetics with a calmodulin-binding target, and control rates of conformational transition. Most notably, these effects are determined by the size and concentration of the crowding reagent indicating that the composition of the environment differentially tunes CaM structure, dynamics, and target association kinetics. Our results are shown in a general kinetic framework where there are consequences for target selectivity and cellular signaling.