A Fluorescence-Based Investigation into the Mechanisms of Biomimetic-Membrane Component Translocation

Restricted (Penn State Only)
- Author:
- Liu, Chang
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 01, 2023
- Committee Members:
- Paul Cremer, Chair & Dissertation Advisor
William Hancock, Outside Unit & Field Member
Tae-Hee Lee, Major Field Member
Lauren Zarzar, Major Field Member
Philip Bevilacqua, Program Head/Chair - Keywords:
- chemotaxis
lipid flip-flop
membrane component translocation
microfluidics
lipid bilayer - Abstract:
- Herein, the translocation of biomolecules, from lipids to proteins, on biomimetic membrane system supported lipid bilayers (SLBs), are investigated by means of fluorescence-based assays. The biomolecules within the lipid bilayer membrane can translocate in two directions: interleaflet translocation from one lipid bilayer leaflet to another and lateral translocation within the same leaflet. In the first part of the thesis, the interleaflet translocation of lipids, also known as “flip-flop”, is shown to occur at defect sites within the SLB. The study employs fluorescence recovery after photobleaching (FRAP) to investigate the subsequential immobilization of lipids due to attractive hydrogen bonding interactions. A novel bioanalytical assay is also developed to decouple the two leaflets of the SLB and directly characterize the distribution of specific lipids within the two leaflets. We reveal that the outcome of the lipid “flip-flop” and immobilization is influenced by repulsive electrostatic interactions and attractive hydrogen bonding interactions, which can be tuned by various factors, including lipid headgroup structure, temperature, pH, ionic strength, and substrate materials. In the second part of the thesis, the study employs SLB platforms within microfluidics to explore the in-plane lateral translocation of model membrane-bound proteins, such as avidin, by steady-state and time-resolved analysis with fluorescence microscopy. It is revealed that these biomacromolecules exhibit directional lateral translocation, named in-plane chemotaxis, when subject to a concentration gradient of ions. Membrane-bound proteins can undergo both positive and negative movement, depending on the identity and concentration of ions. This two-dimensional migration arises from multiple driving forces, including electrostatic screening, diffusiophoresis, diffusioosmosis, and differences in protein-protein interactions (PPIs) as a function of position along the gradient. These chemotaxis mechanisms may play a role in the lateral trafficking of membrane proteins along the surface of living cells. Both peripheral and integral membrane proteins may exploit transient lateral salt concentration gradients to undergo chemotaxis and assist in assembling and disassembling protein complexes. Moreover, in-plane chemotaxis provides a novel, reversible method for reorganizing lipid membranes and performing chromatographic separations.