I) Hv1, LBP and LCF reconstitution in Liposomes II) Signed, Sealed, and Delivered: CPP Technology Delivers Active Protein Cargo
Date of Award
Master of Science in Chemical Sciences (MSCB)
Dr. Susan M.E. Smith
Dr. Jonathan McMurry
Dr. Christopher Alexander
The first half of this thesis focuses on bioluminescence within the Lingulodinium polyedrum dinoflagellates. In this species, the organelle termed “scintillon” houses the components involved in bioluminescence: Luciferin Binding Protein (LBP), Luciferase (LCF), voltage-gated proton channel (Hv1), as well as the substrate Luciferin (LH2). Bioluminescence is triggered by an internal drop in pH of the scintillon initiated by the voltage activation of Hv1 in the membrane. The first focus of this study is to use a synthetic scintillon—a liposome that contains the purified proteins and substrate—to mimic the observed system. Liposomes containing LCF, LBP, and LH2 had little activity compared to mixtures of the free components, suggesting that the liposome reconstitution did not replicate conditions in the naturally occurring scintillon. The second aim was to use FRET to determine the conformational change within the 4th transmembrane helix of the Hv1 protein. Active Hv1 was achieved with two separate protein preparations as shown by quenching of the fluorescent dye ACMA after a valinomycin-induced membrane potential. Technical difficulties prevented further liposome preparations containing active Hv1. The second half of this thesis focuses on cell penetrating peptide (CPP) technology. TaT-CaM is a recently developed CPP that crosses cellular membranes and carries a wide variety of proteins. This study focuses on post-delivery effects of catalase, a peroxidase protein that breaks down H2O2, as proof of concept to show that delivered proteins retain activity after penetration. Quantitation of fluorescence using confocal microscopy showed that TaT-CaM delivered catalase penetrates cells and correctly traffics to the peroxisome. Three activity assays with distinct underlying mechanisms determined catalase to be active post penetration. Although the statistical significance is p~0.06, cells with delivered catalase have a small, repeatable protection against H2O2 toxicity.