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    Studies of Ligand-Receptor Pairs Utilizing Polymerized Planar Supported Lipid Bilayers

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    Author
    Liang, Boying
    Issue Date
    2013
    Keywords
    AFM
    ligand-receptor
    lipid
    MALDI MS
    TIRF
    Chemistry
    affinity capture platform
    Advisor
    Saavedra, Steven S.
    
    Metadata
    Show full item record
    Publisher
    The University of Arizona.
    Rights
    Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
    Embargo
    Release 20-Dec-2014
    Abstract
    Artificial membranes composed of natural lipids are not stable when exposed to air/vacuum, surfactant, organic solvent, etc. Polymerizable lipids provide an opportunity to broaden the use of lipid membranes to study ligand-receptor pairs under harsh experimental conditions. This dissertation presents the utilization of polymerizable lipids in matrix assisted laser desorption and ionization-mass spectrometry (MALDI-TOF MS) for analysis of ligands bound to membrane receptors. This platform may be applied to rapid drug-screening for membrane receptors including transmembrane proteins. Bacterial toxins and their membrane receptors were used as model ligand-receptor pairs to demonstrate the feasibility of using polymerizable lipids to detect and identify ligands by MALDI-TOF MS. Cholera toxin B (CTB) was successfully detected bound to polymerized lipid membranes with incorporation of its membrane receptor, GM1, while no CTB was detected in non-polymerizable lipid membranes. This affinity capture platform based on poly(lipid) showed a high resistance to interferences. On-plate digestion of bound CTB was performed and 57% amino acid sequence coverage was achieved. Total internal reflection fluorescence microscopy (TIRF-M) was applied to compare CTB-GM1 binding affinity in polymerized and unpolymerized membranes. Under a static flow system, the binding between CTB and GM1 was found to be stronger in polymerized membranes than other membranes. However, the ligand concentration under a static flow system is not in excess and the apparent binding affinity is likely to be significantly different than the true value. The true binding affinity can be approached under a continuous flow system, however equilibration time was found to be too long to address experimentally. Membrane fluidity, which may be required to maintain the membrane receptor activity, is suppressed in poly(lipid) membranes compared to unpolymerized membranes. In order to maintain fluidity, a non-polymerizable lipid was mixed into a polymerized lipid. Fluorescence recovery after photobleaching (FRAP) data showed that fluidity of membrane composed of the mixed lipid was maintained compared to pure poly(lipid). Phase segregation of polymerized lipid and non-polymerizable lipid was detected by atomic force microscopy (AFM). CTB bound to GM1 in mixed lipid membranes was detected by MALDI-MS, indicating the mixed lipid membranes retain stability under MALDI-MS analysis conditions.
    Type
    text
    Electronic Dissertation
    Degree Name
    Ph.D.
    Degree Level
    doctoral
    Degree Program
    Graduate College
    Chemistry and Biochemistry
    Degree Grantor
    University of Arizona
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