Insights into Desiccation Tolerance: Properties of Late Embryogenesis Abundant Proteins from Embryos of Artemia franciscana
Doctor of Philosophy (PhD)
LEA proteins are a family of intrinsically disordered proteins that are expressed in various life stages of anhydrobiotic organisms and have been strongly associated with survival during water stress. The brine shrimp Artemia franciscana is the only known anhydrobiotic animal that expresses LEA proteins from Groups 1, 3, and 6. Here, I report that AfrLEA6, a novel Group 6 LEA protein, is most highly expressed in embryos during diapause and decreases throughout pre-emergence development. Notably, there is an acute drop in expression upon termination of the diapause state and the titer of AfrLEA6 during diapause is 10-fold lower than values reported for Group 3 LEA proteins in A. franciscana. Investigations of the secondary structure of AfrLEA6 support classification as an intrinsically disordered protein. Drying the protein or exposure to sodium dodecyl sulfate (SDS) or trifluoroethanol (TFE) promote a large gain in secondary structure of AfrLEA6, predominated by α-helix and exhibiting minimal β-sheet structure. Physiological concentrations of trehalose,a non-reducing disaccharide known to drive protein folding, do not affect the circular dichroism spectra of AfrLEA6, AfrLEA2, or AfrLEA3m in the aqueous state. Furthermore, AfrLEA6 protects enzymes and liposomes from damage during drying. AfrLEA6 protects cytoplasmic enzymes lactate dehydrogenase and phosphofructokinase but not the mitochondrial enzyme citrate synthase; in some cases, the addition of trehalose improves protection. AfrLEA6 weakly stabilizes liposomes simulating the inner leaflet of the plasma membrane, outer mitochondrial membrane, or inner mitochondrial membrane during drying; overall protection is less than with Group 3 LEA proteins. Results of immunohistochemistry support a cytoplasmic localization of AfrLEA6. Finally, an investigation into the structure and function of AfrLEA2 at intermediate water contents reveals a protective role for LEA proteins across a range of hydration states. The ability of AfrLEA2 to stabilize PFK during drying coincides with the gain of α-helix secondary structure as the relative dryness of the sample increases. These findings underscore the concept that multiple LEA proteins exist at different amounts and within different cellular compartments within a single organism and may act together to protect the organism during water stress.
LeBlanc, Blase Matthew, "Insights into Desiccation Tolerance: Properties of Late Embryogenesis Abundant Proteins from Embryos of Artemia franciscana" (2020). LSU Doctoral Dissertations. 5269.
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