Eye lens β-crystallins are predicted by native ion mobility-mass spectrometry and computations to form compact higher-ordered heterooligomers.

Eye lens α- and β-/γ-crystallin proteins are not replaced after fiber cell denucleation and maintain lens transparency and refractive properties. The exceptionally high (∼400-500 mg/mL) concentration of crystallins in mature lens tissue and multiple other factors impede precise characterization of β-crystallin interactions, oligomer composition, size, and topology. Native ion mobility-mass spectrometry is used here to probe β-crystallin association and provide insight into homo- and heterooligomerization kinetics for these proteins. These experiments include separation and characterization of higher-order β-crystallin oligomers and illustrate the unique advantages of native IM-MS. Recombinantly expressed βB1, βB2, and βA3 isoforms are found to have different homodimerization propensities, and only βA3 forms larger homooligomers. Heterodimerization of βB2 with βA3 occurs ∼3 times as fast as that of βB1 with βA3, and βB1 and βB2 heterodimerize less readily. Ion mobility experiments, molecular dynamics simulations, and PISA analysis together reveal that observed oligomers are consistent with predominantly compact, ring-like topologies.