Lens transparency is achieved by maintain structural regularity of cytoplasmic proteins and uniform alignment of fiber cell membranes. Light scattering is minimized of these two levels of structural regularity. Age and cataractous dependent changes in proteins and membranes, in the interaction properties or in structures, will alter structural regularity and cause lens turbidity. Protein concentrations in the lens are, very high and they interact with each other. This interaction provides structural regularity. In contrast, the interaction leading to high molecular weight (HMW) aggregate disrupts the structural regularity. The mechanism of both interactions are not well understood. The formation of HMW aggregate appears to be initiated by protein unfolding, which in turn is caused by post-translational modifications. The process appears to be in the order; post-translational modifications leads to unfolding leads to aggregation leads to insolubilization. The aggregation could be either a protein-protein or a protein-membrane interaction. These events finally lead to lens opacity. It is unknown, however, how the post-translational modifications unfold protein, and how the unfolded protein becomes susceptible to aggregation. It also needs to be established how the aggregated and insoluble proteins become so effective in scattering light in the opaque lens. In this proposed study, the mechanism of the normal protein interactions will be studied first, and then the mechanism in which they are disrupted and replaced by HMW aggregation. The techniques to be used include fluorescence, circular dichroism and NMR. It is not only possible to -study the protein unfolding process, but also structural changes in the aggregated and insoluble proteins. The latter one appears to be an important contributor to light scattering and could be studied by front surface fluorescence and solid-state NMR. Another important factor for lens opacification is the change in membranes. An increased membrane rigidity not only affects the biological functions, but also changes the physical properties such as the refractive index. Measuring fluorescence anisotropy and microviscosity will be done to understand the effect of increased cholesterol and associated-crystallins. The significance of this proposed study is that it puts some emphasis on the study of concentrated protein solution,.protein powder and lens gel. Studies progressing from dilute solution to concentrated solution, and finally to lens, may provide information more relevant to what really occurs in vivo.
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