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

Persistent Link:
http://hdl.handle.net/10150/311232
Title:
Studies of Ligand-Receptor Pairs Utilizing Polymerized Planar Supported Lipid Bilayers
Author:
Liang, Boying
Issue Date:
2013
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
Keywords:
AFM; ligand-receptor; lipid; MALDI MS; TIRF; Chemistry; affinity capture platform
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry and Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Saavedra, Steven S.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleStudies of Ligand-Receptor Pairs Utilizing Polymerized Planar Supported Lipid Bilayersen_US
dc.creatorLiang, Boyingen_US
dc.contributor.authorLiang, Boyingen_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © 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.en_US
dc.description.releaseRelease 20-Dec-2014en_US
dc.description.abstractArtificial 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.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectAFMen_US
dc.subjectligand-receptoren_US
dc.subjectlipiden_US
dc.subjectMALDI MSen_US
dc.subjectTIRFen_US
dc.subjectChemistryen_US
dc.subjectaffinity capture platformen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistry and Biochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorSaavedra, Steven S.en_US
dc.contributor.committeememberSaavedra, Steven S.en_US
dc.contributor.committeememberAspinwall, Craig A.en_US
dc.contributor.committeememberHeien, Michael L.en_US
dc.contributor.committeememberGhosh, Indraneelen_US
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