Our main objective is to understand the molecular mechanisms by which proteins that are normally soluble, undergo transformations that lead to lowered solubility and to disease. For several years we have been determining the mechanisms by which genetic mutations and chemical modifications of the crystallin proteins in the eye lens, lead to a variety of cataracts (genetic and age-related). Based on available genetic and epidemiological data, we have examined the mutant proteins associated with cataract and proposed molecular mechanisms which relate the observed pathology (or phenotype) to the altered molecular interactions caused by the mutation.
The strategy involves expressing native and mutant crystallins in E. coli and comparing their physico-chemical properties in solution. Our in-vitro studies have resulted in significant, novel findings: We found that unlike the native proteins which are highly soluble, the mutant proteins undergo a number of phase transformations that reduce their solubility and result in the formation of a variety of condensed-phases (such as protein-rich and protein-poor liquid phases, protein crystals, covalent and reversible aggregates, gels and fibers). These condensed phases, formed in solution, are responsible for the light scattering and opacity that lead to cataract. Despite these distinct phase changes however, the 3-D x-ray crystal structure as well as the solution conformation of the mutant proteins, remain essentially intact. Nevertheless, we plan to examine if small, localized structural perturbations occur in the protein structure due to the mutation or modification, and contribute to the condensation of the proteins. We will investigate this possibility using high-resolution structural, and targeted spectroscopic and chemical studies of the native and mutant proteins in the next phase of this work. This work will be facilitated by the installation of high-end static and time-resolved spectrofluorometers in the Life Sciences Research Building in the near future, as part of the core facilities. A 700 MHz NMR spectrometer is already available on the premises for this purpose.
A key aspect of our work involves using Raman spectroscopy and imaging to compare intact normal and cataractous lens tissue. Lens epithelial and fiber cells will also be examined using optical imaging and micro-spectrophotometry. A state-of-the-art Raman microscope will be purchased for these studies in the near future.
We have also hypothesized that, as in the case of cataract, condensation of mutants of the protein myocilin, plays a significant role in some forms of primary open-angle glaucoma. Preliminary data suggest that some glaucoma-associated myocilin mutants undergo phase changes similar to that observed in various forms of cataract. Examination of these mutants in solution and in mammalian cells in culture, will constitute a major part of our work on glaucoma.