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M. J. Clarke, J. Carver, P. Griffiths, J. Harding, K. M. Meek, P. Timmins, J. W. Regini; Investigation of Target Protein Binding to Bovine Lens Alpha-Crystallin by Small-Angle Neutron Scattering. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1635.
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© ARVO (1962-2015); The Authors (2016-present)
-crystallin is the main constituent protein in the crystalline lens. Presently, the structural relationship between -crystallin and its target proteins during chaperone function is poorly understood. Experiments were undertaken to investigate the interaction of a target protein with -crystallin.
Small-Angle Neutron Scattering (SANS) and contrast variation were used in conjunction with isotopic substitution of expressed γE-crystallin to quantify the radius of gyration (Rg is a measure related to the size of proteins and complexes) of the proteins and their binary complexes in solution. This allows investigation into the interactions between the binary mixtures of the target protein and bovine -crystallin. Scattering intensity profiles were obtained from hydrogenous -crystallin (H), deuterated γE-crystallin (γD), and hydrogenous γE-crystallin (γH) individually and in binary mixtures. SANS experiments were carried out at 65oC to investigate the effect of thermal stress.
Rg for H in D2O was consistent with previously published Small Angle X-Ray Scattering and SANS measurements at (62±2Å to 68±2Å over 40 minutes), with no evidence of protein aggregation. In the contrast variation experiment, it is possible to distinguish one protein from another by virtue of the fact that very little scattering is seen from γD in D2O or from H in H2O. Thus, scattering from the H:γD complex in H2O arises predominantly from the γD component. Data shows that scattering from the thermally stressed H:γD complex in H2O changes with time and that this is associated with aggregation. In light of kinetic studies of protein chaperone action, the scattering was followed for 40 min at 65oC to probe the evolution of the aggregation size/shape. Rg increases from 34±3Å to 50±3Å over this period, unlike Rg for the complex of H:γD in D2O which measures 60Å±3Å to 68±3Å over 40 minutes and was found to be similar to H in D2O at 62±2Å to 68±2Å over 40 minutes.
The Rg of the H:γD complex in H2O (which ranges from 34±3Å to 50±3Å over 40 minutes) is considerably smaller than that of -crystallin alone in D2O (62±2Å to 68±2Å). This lends weight to the hypothesis that γE-crystallin sits in a central cavity of the -crystallin.
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