Date of Award

1-1-2013

Document Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

First Advisor

Michelle K. Knowles

Keywords

Biostatistics, C-reactive protein, Curvature Sensing, Diffusion, Lipids, Supported lipid bilayer

Abstract

The physical structure of cellular membranes plays a critical role in lipid and protein sorting. A novel biosensor was developed to probe the influence of curvature on sorting. This biosensor mimics large, two-dimensional membranes in dynamic equilibrium, achieves high spatial resolution between curvature and molecules of interest, and has high sensitivity, enough for single particle detection. The biosensor consists of continuous supported lipid bilayer formed over nanoparticles (40 to 200 nm diameter) deposited on a glass substrate. The nanoparticles determine the extent of curvature. This biosensor is the first to observe large-scale 2-dimensional diffusion of biomolecules on a supported lipid bilayer with small radii of curvature in equilibrium with flat areas of fluid bilayer. This will allow correlation between protein function and the physical shape of a membrane. Fluorescence microscopy was used to quantify spatial sorting of lipids and a protein relevant to cardiovascular disease, C-reactive protein (CRP). Two lipids, fluorescein labeled hexadecanoic acid and 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, sense curvature by accumulating in areas over nanoparticles, and both are able to laterally exchange with surrounding lipids. Dynamic fluidity of the bilayer was assessed using fluorescence recovery after photobleaching. Lipids directly at sites of curvature recover more slowly than lipids over flat sections. The spatial distribution of CRP was also assessed. Curvature sensing of CRP is isoform dependent where native CRP does not sense curvature and modified CRP does sense curvature. Finally, we show that a ribonucleic acid aptamer will bind specifically to modified CRP and not native CRP, enabling isoform specific studies of CRP to be conducted.

Provenance

Recieved from ProQuest

Rights holder

Joshua Black

File size

130 p.

File format

application/pdf

Language

en

Discipline

Chemistry, Biophysics

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