Manipulating polydispersity of lens β-crystallins using divalent cations demonstrates evidence of calcium regulation

One common feature conserved across living systems is the high concentration of crystallin proteins packed within the eye lens. Of the three most common vertebrate subtypes, β-crystallins exhibit the widest degree of polydispersity due to their complex multimerization properties in situ. While the structural complexities of crystallins is one factor that aids in preventing phase-separation within the protein-dense environment, properties which enable vision, it is unclear whether the polydispersity of β-crystallins provides additional function or is a redundant feature in the lens. One proposed function for β-crystallins is calcium-buffering. The lens needs to control calcium to prevent rampant proteolysis by calpains, while balancing protein-protein interactions to maintain tissue transparency. Our combined results suggest that protein-protein and protein-cation interactions are in competition in lens β-crystallins. We assayed hydrodynamic behavior and assembly mechanics of crystallins in different salts and observed altered protein polydispersity in vitro. We used size-exclusion chromatography together with dynamic light scattering and LC-MS/MS to show how divalent cations dissociate β-crystallin oligomers, reducing polydispersity, and shifting protein surface charge – properties that can be reversed when the salts are removed. While the direct, physiological relevance of these divalent cations in the lens is still under investigation, our results support that specific isoforms contribute to a dynamic equilibrium and inherent polydispersity of β-crystallin as an important feature for two aspects of lens function: calcium-regulation and microstructure. The cation-regulating mechanisms of β-crystallin outlined by our results are expected to contribute to the long-lived stability and transparency of the lens.