George B. Benedek Group Homepage

Physical and Chemical Basis for Lens Opacity

Personnel: Jennifer J. Mc Manus, Aleksey Lomakin, Ajay K. Pande, Olutayo Ogun, Jayanti Pande, George B. Benedek

Our long-term objective is to identify the molecular factors responsible for light scattering and opacification of the lens. Light scattering can be produced in the lens by the formation of protein aggregates, the condensation of proteins into coexisting protein-rich and protein-poor liquid phases and the condensation of proteins into crystalline solid phases. We have identified various molecular mechanisms that produce such condensates, and thereby result in light scattering and opacification in gamma crystallin solutions in-vitro. Some of these mechanisms are now known to be responsible for several human genetic cataracts. Our current interests are in Gamma and Beta crystallins and their mixtures.

Point mutations in Human Gamma-D Crystallin

Gamma D Crystallin is a major component of the human eye lens. Point mutations at various sites on HGD are associated with different cataract phenotypes [1]. We have shown that Site 23 of HGD is of particular interest. Replacement of Proline at site 23, by a number of different amino acid residues (Thr, Ser, Val), reduces the solubility of the protein. Furthermore, these mutants display inverted solubility, with the protein being most soluble at lower temperatures. This marked change due to point mutatations at this single site is significant and striking and thus merits further and more detailed investigation.

We have focused on the Pro23 to Val mutation of HGD in this study. While all of the mutants we have investigated showed this inverted solubility, P23V is soluble over a wide temperature and concentration range (in contrast to the cataract associated P23T mutant), making it experimentally most accessible for detailed investigation. The native-like solubility of P23V coupled with its strong similarity to P23T in forming condensed phases over a wide concentration range, and within a workable temperature region, gives us the advantage of a wide range of experimental conditions to examine the solution properties of this mutant in detail. In cataract disease, the Pro23 to Thr mutation leads to opacifciation in the eye lens due to the formation of aggregates. The P23T mutation is associated with many cataract phenotypes; Coralliform, Cerulean and Fasciculiform. We believe that understanding the importance of site 23 in HGD will give us a better understanding of the of the mechanisms leading to opacification in the eye lens due to mutations at this site.

We have mapped the phase diagram to determine the nature of the condensed phases formed by P23V. We have also used Quasielastic Light Scattering to investigate the diffusive behavior of the protein and to understand the kinetics leading to both aggregation and crystallization.