Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Tobias Baumgart

Abstract

Biological membranes are constantly being remodeled with high spatial and temporal accuracy, which is essential during various cellular processes, including cell trafficking, cell signaling, and the generation and maintenance of organelles. Critical to these cellular processes is the Bin/Amphiphysin/Rvs (BAR) domain superfamily, whose crescent-shaped scaffold allows them to sense and induce membrane curvature. Membrane curvature generation can occur through one or more of at least four mechanisms that are not mutually exclusive, including scaffolding, hydrophobic insertion, oligomerization, and crowding. One BAR domain class, N-BAR, contains an N-terminal amphipathic helix. The amphipathic helix (AH) motif is a common protein folding motif. Many AHs are disordered in solution and will adopt an α-helical structure upon membrane association.

α-Synuclein is an intrinsically disordered protein in solution but will form a uniquely long AH upon association with anionic membranes. It is known that α-synuclein significantly expands the membrane proportionally to α-synuclein’s membrane binding density. Firstly, we hypothesized that membrane-expanding proteins might adjust their binding in response to lateral membrane tension. To test this hypothesis, we used model membranes in the form of Giant Unilamellar Vesicles. Membrane tension was altered by osmotic stress and via micropipette aspiration. I reveal that α-synuclein can change its membrane binding density in response to osmotic membrane tension changes. Our observations reveal a mechanism by which different types of peripheral membrane proteins might change their binding during processes such as endo- and exocytosis that involve membrane tension changes.

Secondly, I develop a model for the molecular basis of F-BAR protein, FBP17, as a plasma membrane tension sensor. Since the molecular protein density is intricately connected to membrane tension, I assess FBP17’s curvature sensing and generation ability on both tensionless and tense membranes. I demonstrate that at high protein densities FBP17 can form an immobile protein coat on the membrane, which may prevent curvature generation. Furthermore, I show that cholesterol can facilitate FBP17’s curvature generation capacity and that it can accentuate FBP17’s role as a tension sensing protein.

Thirdly, I ask to what extent molecular properties of the H0 helix of N-BAR endophilin affect membrane targeting behavior. Five different endophilin isoforms, grouped into A and B subclasses, have been described to differ in their subcellular localization and function. I determine that the presence of anionic phospholipids is critical for all H0 helices partitioning into membranes. Membrane partitioning is found to be sensitive to variations in membrane complexity. The different H0 helix subfamilies display different membrane binding preferences that are influenced by both electrostatic interactions and hydrophobic contributions.

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