Project Summary The vertebrate eye lens is made up of concentrated, highly refractive proteins called crystallins, which enable it to form images by focusing light on the retina. Most studies of crystallins focus on their ability to remain stable and soluble for decades in the absence of protein turnover in the lens. Refractivity is equally important to crystallin functionality, but is less well understood. This project seeks to elucidate how crystallins provide focusing power both as individual proteins and as a network of intermolecular interactions in the crowded environment of the lens. Our preliminary work has shown that amino acid composition alone does not determine crystallin refractiv- ity: three-dimensional structure and interactions with water on the protein surface are also critical. We will build on this work by measuring the refractive index increment for lens crystallins from humans and model organisms and relating the results to spatial interactions among polarizable side-chain moieties. In particular, we propose to investigate cataract-related and engineered crystallin variants in order to test hypotheses about how structure impacts refractivity. We will measure refractivity first in concentrated solutions mimicking the crowded cellular milieu, and then in whole lenses from model organisms in order to set the stage for future studies on human lenses. This will enhance the current model of protein refractivity, which is mostly based on measurements in dilute solution. On the level of the whole lens, we will develop novel methodologies to specifically visualize the refractive index distribution in space. The long-term goal of this research is to bridge the gap between interactions of light with individual crystallin molecules and the spatial arrangement of proteins that generates the refractive index gradient of the vertebrate lens. In the future, the knowledge gained will provide insight into healthy lens function and guide the design of improved artificial lens materials.