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

Abstract

Crystallins comprise the protein-rich tissue of 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 polydispersity enables precise packing densities across the concentration gradient of the lens for vision, it is unclear why there is such a high degree of structural complexity within the β-crystallin subtype and what the role of this feature is in the lens. To investigate this, we first characterized β-crystallin polydispersity and then established a method to dynamically disrupt it in a process that is dependent on isoform composition and the presence of divalent cationic salts (CaCl 2 or MgCl 2 ). We used size-exclusion chromatography together with dynamic light scattering and mass spectrometry to show how high concentrations of divalent cations dissociate β-crystallin oligomers, reduce polydispersity, and shift the overall protein surface charge—properties that can be reversed when 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 of β-crystallin modulate polydispersity through multiple chemical equilibria and that this native state is disrupted by cation binding. This dynamic process may be essential to facilitating the molecular packing and optical function of the lens.

Design and Characterization of Model Systems that Promote and Disrupt Transparency of Vertebrate Crystallins In Vitro

“…[ 68 , 69 ] However, this is not expected to significantly affect the structure or network interactions of lens crystallins, but could possibly affect the chaperone efficacy of α‐crystallin. [ 46 , 70 ] Because turbidity onset occurred almost immediately after deposition of the PEG, it was difficult to capture a nucleation or lag phase in the kinetics; however, we did observe an increased slope, approaching a plateau which is common among other aggregation mechanisms. [ 71 , 72 , 73 ] To investigate whether this turbidity could be reversed when the buffer was exchanged from PEG to 1x TBS containing EDTA to disrupt the alginate matrix, we observed no changes in turbidity, suggesting crystallin condensation was irreversible within the matrix (Figure S8 , Supporting Information).…”

“…In these simulations, β‐crystallin is more representative of β H , which is the most prominent oligomeric form of β‐crystallin in the lens. [ 46 ] Moreover, there were leftward shifts in both the troughs and peaks of the RDF at ϕ = 0.4 compared to the regular periodicity in monodisperse α‐crystallins, where the peaks appeared at regular intervals of r = 1, 2, and 3. Polydispersity diminished the long‐range order present in the α‐crystallin only system.…”

“…[ 41 , 42 ] Hard sphere simulations of α‐crystallins and β‐crystallins were previously used to describe the conditions needed to induce dynamical arrest, i.e., when particles effectively stop motion, observed in experimental studies. [ 43 , 44 , 45 ] To build on this, we utilized the concentration ratios of crystallins that were previously determined in bovine lens [ 46 ] along with average diameters measured with DLS (Figure S2 , Supporting Information) to construct the particle compositions in the simulations.…”

“…[ 43 , 44 , 47 , 48 ] We suspect this long‐range ordering may decrease the transparency of the system, as it favors a more rigid structure with fewer short‐range interactions that are necessary for transparency. The characteristic periodic peaks in the RDF and S( k ) of α‐crystallins were immediately diminished in a polydisperse system consisting of α‐, β‐, and γ‐crystallin particles represented with a mole fraction of 0.05:0.20:0.75 (α:β:γ), which is calculated from the experimental mass ratios, [ 46 ] averaged across the lens and converted to moles using the estimated molecular weights based on known protein standards (BioRad) of 680, 180, and 21 kDa for α‐, β‐, and γ‐crystallin respectively (Figure 2b ). In these simulations, β‐crystallin is more representative of β H , which is the most prominent oligomeric form of β‐crystallin in the lens.…”

“…[ 50 ] Since the orientation of water at the inner double layer results in a positive charge, [ 52 , 53 ] differences in charge can have an effect on interfacial behavior. [ 50 , 54 ] And while all crystallins are expected to be negatively‐charged at a neutral pH, it is possible that even the slight differences in surface charge of β H and β L , with isoelectric points of 5.21 and 4.97 respectively, [ 46 ] are enough to elicit differences in interfacial behavior.…”