Doctoral Dissertations

Date of Award

12-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemical Engineering

Major Professor

Eric T. Boder

Committee Members

Steven M. Abel, Paul Dalhaimer, Elias Fernandez

Abstract

Membrane fusion proteins are expressed by many viruses and play a crucial role in fusing two distinct membranes. Hemagglutinin (HA), the viral fusion protein of influenza, catalyzes fusion of the viral and target membrane by undergoing a conformational change triggered by a lowered pH of the endosome. The rearrangement, which is essential for fusion activity, exposes a highly conserved fusion peptide region. An engineered HA protein, with an altered fusion peptide region, has a high affinity for the fluorescent ligand, FlAsH. Using the engineered sensing HA, which allows real time monitoring of open state, we probe for kinetics of the conformational change.We report that the engineered sensing HA, with an altered fusion peptide region that was designed to bind FlAsH after activation, shows induction by FlAsH binding at pH 7.0. The engineered HA shows activation of the fusion associated conformational change induced by alternative environmental stimuli compared to the wild-type protein. We suggest a novel alternative pathway to activation through binding the fusion peptide region which could impact the treatment of many enveloped viruses. We demonstrate that the engineered sensing HA co-expressed with fusion capable wild-type HA create modular systems that drive fusion triggered by ligand binding.We further investigate the role of membrane properties in the activation of HA and the fusion induced by the viral protein. We show that the depletion of cholesterol leads to a significant delay in the kinetics of conformational activation and the extent of fusion. We also examine peptides that alter the membrane and show that disruption leads to the conformational change required for fusion. These results indicate that further studies of the HA behavior in synthetic membranes with defined phospholipids would be beneficial.To design new alternative environmental stimuli, we probed the entire HA gene for potential mutagenesis sites. Creating random mutant libraries and screening for desired phenotypes we have assessed the amino acids that can tolerate mutations with the help of next generation sequencing. With a landscape of tolerated mutational positions we can guide future rational design when trying to engineer the conformational change induced by a new exciting environmental stimulus.

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