Small molecule stabilization of A149P-aldolase, the most prevalent form of aldolase B associated with hereditary fructose intolerance

Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Hereditary fructose intolerance (HFI) is a disease of carbohydrate metabolism caused by aldolase B deficiency. The most common HFI mutation is an alanine to proline substitution at amino acid position 149 (A 149P or APaldolase). AP-aldolase has greater than 200-fold reduced activity as compared to wild-type aldolase B, and is structurally unstable. The hypothesis that these defects in AP-aldolase can be alleviated by small molecules is tested using osmolytes. Osmolytes are small molecules that protect proteins from osmotic stress. The analysis of eleven osmolytes revealed that glycine, betaine, and sarcosine, all zwitterions at molar concentrations, protect AP-aldolase structure and activity from thermal inactivation. Osmolyte classes are also tested for stabilization of two pairs of proteins (different vs. same fold ; hemoglobin/green fluorescent protein and triose phosphate isomerase [TIM]/aldolase B). The effectiveness of osmolytes at protein stabilization varies from protein to protein, even for aldolase and TIM , which have the same a/~ barrel fold. However, the three aldolase isozymes (A, B, and C) all show thermal protection using glycine. These data are inconsistent with two previously proposed models for osmolyte induced protein stabilization, namely the transfer free-energy model and an excluded-volume model. The mechanism of osmolyte-mediated protein stabilization for AP-aldolase is tested with zwitterions that have decreasing fractional polar surface area and increasing excluded volume. The results suggest that osmolyte-mediated AP-aldolase stabilization is not due to either previously proposed mechanism, but rather involves direct binding of osmolyte to AP-aldolase in its native conformation, which is supported by a binding isotherm of glycine concentration versus thermal stabilization. Taken together, these results indicate that the native state of a protein plays a major role in determining the effectiveness of a given osmolyte-protein pair. This model provides a foundation for developing stabilizing compounds as therapeutics for HFI and other protein conformational diseases. Towards this end, stabilizing ligands are investigated by computational design and chemical library screening. Small molecules binding to AP-aldolase are predicted using computational solvent mapping. Computationally predicted compounds, along with thousands of random compounds, are screened for their ability to stabilize AP-aldolase activity. The implications of these studies are discussed.2031-01-0