Design of supramolecular complexes for stability enhancement of membrane protein-based biomaterials.
Item
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Title
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Design of supramolecular complexes for stability enhancement of membrane protein-based biomaterials.
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Identifier
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AAI3187458
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identifier
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3187458
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Creator
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Sharma, Manoj K.
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Contributor
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Adviser: M. Lane Gilchrist
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Date
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2005
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Language
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English
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Publisher
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City University of New York.
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Subject
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Engineering, Chemical
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Abstract
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Membrane proteins are some of the most sophisticated molecules found in nature. Due to their extraordinary molecular recognition capabilities they represent a vast source of functional building blocks for potential use in sensing and drug screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biologically-based materials from these molecules. In general, the purification protocols remove the stabilizing native lipid structures found in the cellular environment. Our research is focused on a biomimetic approach to reintroduce the supporting structures of membrane proteins. The goal is to fabricate robust assemblies for membrane proteins that can withstand analyte flows and processing conditions (e.g., temperature, high ionic strength, organic solvents etc.) while retaining their functionality.;Reconstitution of membrane proteins into the bilayer of lipid vesicles is a standard approach to recreate their microenvironment after isolation from a native cellular source. However, the inherent delicate nature of the lipid membrane limits the applicability of the liposomes. In the current study we have designed lipid bilayers internally anchored to a solid microparticle interface through integral tethering molecules. In earlier designs of tether-supported membranes, primarily single lipid moieties at the end of the tethers have been used to anchor the membranes to solid supports. We utilized bacteriorhodopsin (bR), a transmembrane protein, as a tethering molecule as it is expected to impart more stability to the tethered bilayer compared to a lipid tether. We conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. The conjugates were characterized using SDS-PAGE and MALDI-MS.;A bottom-up, silane-based method was used to functionalize the surface of silica microspheres (5-mum) with streptavidin in order to provide bR-PEG 3400-biotin tether anchoring sites for the supported membrane. Tether-supported lipid-bilayer membranes with native-like fluidity were assembled successfully on the functionalized silica microspheres, verified using confocal microscopy. This method is a new platform for the immobilization of active membrane proteins in native lipids, and a gateway to new materials and diagnostics based on membrane protein molecular recognition.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
