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

alpha-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 alpha-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 (gamma E-crystallin) with alpha-crystallin (alpha(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 alpha(H) in D2O incubated at 65 degrees C increased from 69 +/- 3 to 81 +/- 5 angstrom over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated gamma E-crystallin in H2O buffer (gamma E-D/H2O) and hydrogenous gamma E-crystallin in D2O buffer (gamma E-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 gamma E:alpha(H) complexes. The evolution of the aggregation size/shape as an indicator of aH chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 degrees C) for binary mixtures of alpha(H), gamma E-H, and gamma E-D. It was found that Rg(alpha(H):gamma E-D/D2O)>Rg(alpha(H):gamma E-H/D2O)>Rg(alpha(H)/D2O) and that Rg(alpha(H):gamma E-H/ D2O)approximate to Rg(alpha(H)/D2O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that gamma E-crystallin binds in a central cavity of the alpha-crystallin oligomer, during chaperone action. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.