Biostabilization of Lipid Bilayers: Dealing with Water Stress in Embryos of Artemia franciscana

The aim of this dissertation is to investigate stabilization of lipid bilayers during water stress by protectants found in embryos of Artemia franciscana. Two LEA proteins were used: AfrLEA2 and AfrLEA3m. AfrLEA3m was experimentally demonstrated to reside in the matrix. Two detergents were used to differentially solubilize the outer and inner membranes of mitochondria isolated from A. franciscana. Release of AfrLEA3m occurred simultaneously with the release of fumarase, a matrix-resident marker. As a second independent method to corroborate the above findings, I demonstrated that recombinant AfrLEA3m can be imported into mitochondria isolated from rat liver. Molecular modeling of AfrLEA2 and AfrLEA3m revealed structural features that are consistent with amphipathic proteins able to interact with and stabilize cell membranes. The abilities of trehalose and LEA proteins to protect liposomes of various compositions from desiccation-induced damage were evaluated by carboxyfluorescein leakage. AfrLEA2 (cytoplasmic) and AfrLEA3m (mitochondrial) were able to offset damage during drying of liposomes that mimicked the lipid compositions of the inner mitochondrial membrane, outer mitochondrial membrane, and the inner leaflet of the plasma membrane. LEA proteins were more effective than trehalose at preventing desiccation-induced damage when these protectants were confined to one side of the lipid bilayer. When LEA proteins were used in conjunction with trehalose, additive protection was measured in some cases. Little to no additional damage occurred to liposomes dried for one week compared to liposomes dried overnight. The capacity of trehalose and LEA proteins to protect liposomes from freeze-thaw damage was also assessed. Damage to liposomes was less severe after freezing than desiccation. Trehalose provided liposomes with greater protection than LEA proteins from freeze-thaw damage. The greatest stabilization during freezing occurred when trehalose was present on both sides of liposome membranes. Only liposomes mimicking the outer mitochondrial membrane were significantly protected from freeze-thaw damage by LEA proteins. Based on bioenergetic properties assessed by respirometry, the outer membrane of isolated mitochondria (rat liver) remained intact after freezing in 300 mM trehalose solution. Respiratory control ratios were depressed by approximately 30% compared to non-frozen mitochondria, which indicated a limited retention of at least some inner-membrane-dependent properties