Investigation of γE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering

α-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between α-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (γE-crystallin) with α-crystallin (α_H) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the α_H in D₂O incubated at 65 °C increased from 69 ± 3 to 81 ± 5 Å over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated γE-crystallin in H₂O buffer (γE_D/H₂O) and hydrogenous γE-crystallin in D₂O buffer (γE_H/D₂O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate γE:α_H complexes. The evolution of the aggregation size/shape as an indicator of α_H chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 °C) for binary mixtures of α_H, γE_H, and γE_D. It was found that Rg(α_H:γE_D/D₂O) > Rg(α_H:γE_H/D₂O) > Rg(α_H/D₂O) and that Rg(α_H:γE_H/D₂O) ≈ Rg(α_H/D₂O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that γE-crystallin binds in a central cavity of the α-crystallin oligomer, during chaperone action. Crown