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Thesis for Xiaosi Li 08-17-2019.pdf (11.84 MB)

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Lipid-peptide interaction in biological membranes with fluorescence correlation spectroscopy

Li, Xiaosi
http://orcid.org/0000-0002-8220-5952
http://rave.ohiolink.edu/etdc/view?acc_num=akron1564504911054232

Abstract Details

2019, Doctor of Philosophy, University of Akron, Chemistry.
Molecular interactions drive molecular assembly and organization in the plasma membrane. Specific protein-lipid interactions, however, are difficult to resolve. The critical factor in studying these interactions is the ability to determine the dynamics of protein/peptide interactions in plasma membrane. Here we report on a unique approach to investigate these interactions with time-resolved fluorescence spectroscopy. Chapter III focuses on the electrostatic interaction of anionic lipids and peripheral peptides in plasma membrane. The experiments were performed on a model membrane system consisting of a supported lipid bilayer with an asymmetric distribution of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the upper leaflet of the bilayer. The bilayer also contained nickel-chelating lipids that bind to a histidine-tagged peptide of interest. Both the peptides and the anionic lipids were labeled with orthogonal fluorescent probes so that diffusion and binding could be measured with two-color, pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The PIE-FCCS data showed significant lipid-peptide cross-correlation between PIP2 lipids and membrane-bound cationic peptides, and these data provide a direct indication of lipid-peptide binding and complexation. With a two-component diffusion fit, we quantified the degree of lipid-peptide binding, as well as the size of the lipid-peptide complexes. Chapter IV studies the fluorescence lifetime of BODIPY-labeled lipids in two types of model membranes. The fluorescent lifetime of BODIPY-labeled lipids could be altered by molecular-dye interactions, dye aggregations, or interactions of fluorescent molecules. However, few studies examine the factors underlying the effect of fluorescence lifetime of fluorescent-labeled lipid in plasma membrane. One such fluorescent dye now commonly used to examine the lipid dynamics in cellular membrane is a derivative of BODIPY, TopFluor. In this work, we have measured the fluorescence lifetime of TopFluor labeled phosphatidylserine (TopFluor-PS) lipid in two types of model membranes. We have demonstrated that the fluorescence lifetime of TopFluor-PS varies from small unilamellar vesicles (SUVs) to supported lipid bilayers (SLBs). Moreover, the model membrane consisted of a nickel-chelating lipid that can influence the fluorescence lifetime of TopFluor-PS. Furthermore, the binding affinity between anionic lipids and peripheral membrane proteins/peptides were characterized by fluorescence lifetime measurement. Chapter V describes a method for preparing Gram-negative inner membrane and Gram-positive SLBs, including asymmetric SLBs (asy-SLB) that contain a fluorescent tracer only in the upper leaflet of the membrane. Model membranes are a valuable tool to investigate the mechanism of interaction between the antibiotic compound and bacterial membranes. However, the development of supported lipid bilayer (SLB) models for Gram-negative and Gram-positive bacteria is challenging because of the high concentration of charged lipids. We quantified the dynamics of the lipids in these membranes with fluorescence correlation spectroscopy (FCS) and found that lipid diffusion is slower in Gram-negative SLB/asySLB than in Gram-positive SLB/asySLB. Peptide binding to these membranes was also characterized using colistin, a Gram-negative specific antibiotic. Interactions between colistin and membrane lipids phosphatidylethanolamine (TopFluor-PE) or cardiolipin (TopFluor-TOCL) were probed with PIE-FCCS. Low levels of cross-correlation were observed between colistin and TopFluor-PE, but not TopFluor-TOCL. The diffusion coefficient of TopFluor-PE with colistin was lower than TopFluor-TOCL with colistin on the membrane. Together these data indicate that TopFluor-PE exhibited stronger colistin binding behavior than TopFluor-TOCL, consistent with the fact that colistin is a Gram-negative specific bactericide.
Adam W. Smith (Advisor)
Chrys Wesdemiotis (Committee Member)
Aliaksei Boika (Committee Member)
Michael Konopka (Committee Member)
Jie Zheng (Committee Member)
156 p.
Biochemistry; Physical Chemistry

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Citations

  • Li, X. (2019). Lipid-peptide interaction in biological membranes with fluorescence correlation spectroscopy [Doctoral dissertation, University of Akron]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=akron1564504911054232

    APA Style (7th edition)

  • Li, Xiaosi. Lipid-peptide interaction in biological membranes with fluorescence correlation spectroscopy . 2019. University of Akron, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=akron1564504911054232.

    MLA Style (8th edition)

  • Li, Xiaosi. "Lipid-peptide interaction in biological membranes with fluorescence correlation spectroscopy ." Doctoral dissertation, University of Akron, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=akron1564504911054232

    Chicago Manual of Style (17th edition)

akron1564504911054232
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This open access ETD is published by University of Akron and OhioLINK.