Graduate Thesis Or Dissertation
 

Probing Structure, Selectivity, and Orientation of Biomolecules to Cell Membranes by Sum Frequency Generation

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  • Cell membranes act as barriers against unwanted movement of ions and biomolecules and are complex surfaces littered with protein channels, ion channels, cholesterol, and a myriad of different proteins. While this surface is extremely well studied, there is still much we do not know. The work in this dissertation demonstrates how information about the orientation, structure, and selectivity of biomolecules interacting with cell membranes can be characterized by sum frequency generation (SFG) vibrational spectroscopy. The presented work first started with a small protein with a single defined structure and developed into larger more complex proteins as well as nanoparticles. First, we started with a model protein-membrane system for a peptide (WLBU2) that was predicted to have two difference structures, an active 𝛼-helix in the presence of a bacterial membrane and an inert 𝛽-strand when in the presence of a mammalian membrane. A lipid monolayer at the air-water interface was used at the model membrane for observing the peptide interactions. Using SFG, vibrational spectra from amide bonds on the backbone of the protein were collected and resonances assigned to the active 𝛼-helix and inert 𝛽-sheet structure were observed. These experiments demonstrated SFG capability to observe structure and selectivity of a peptide interacting with a model cell membrane. Building on the previous system, the orientation and structure of more complex proteins with multiple secondary structures in their tertiary structures was completed. Three complex C2 domain proteins (otoferlin C2F, dysferlin C2A, and dysferlin C2A D16A) interacting with a model lipid monolayer comprised of phosphatidylserine and phosphatidylcholine lipids were characterized. The amide backbone resonances unique for each secondary structure from individual folding motifs of the proteins result in a complex spectra containing important spectral information. To delineate the protein folding from the experimental SFG spectrum, theoretical spectra calculated form the results of molecular dynamic simulations are contoured to the experimental SFG spectra. This process allows information such as orientation and structure to be assigned to the complex multi-secondary structure C2 domains, providing the first characterization of ferlins directly interacting with a lipid membrane. Lastly, by taking advantage of the SFG ability to detect changes in structure, a shape depended study of nanoparticles interacting with a lipid membrane was conducted. For these experiments nanoparticle functionalization, lipid membrane charge, and relative number of nanoparticles was kept constant such that the only variable tested is shape. By characterizing the nanoparticles from the perspective of the membrane, the result was that differently shaped nanoparticles had a profoundly different impact in the structure of the lipid membrane.
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  • This work was supported by the John C. Erkkila, M.D. Endowment for Health and Human Performance and by NIH National Institute of Deafness and Other Communication Disorders (NIDCD) Grant R01DC014588.
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  • Ongoing Research
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  • 2018-06-01 to 2020-07-02

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