Author: James Eric ShawPublisher:
ISBN: 9780494393802
Category :
Languages : en
Pages : 278
Book Description
Processes occurring at membrane interfaces are important for cellular and biological functions. The inability to readily crystallize membrane proteins for X-ray diffraction, or in solution NMR to study proteins with large molecular weight and to retain all intramolecular structures of the membrane proteins during their solubilisation process by detergents are the key reasons for a scarcity of atomic-resolution membrane protein structures. This has hindered our understanding of the structure-function relationship of membrane proteins. New strategies for studying membrane proteins will provide more insights. In this thesis, we have coupled atomic force microscopy and two fluorescence microscopy techniques---total internal reflectance fluorescence microscopy and confocal laser scanning microscopy, to visualize protein-membrane interactions and dynamics in model membrane systems. To validate our correlated approach, we investigated the partitioning of various lipid fluorescent probes in phase-separated supported planar bilayers. We applied the same methodology in two case studies of peptide-membrane interactions in supported planar bilayers: (1) indolicidin, a cationic 13 amino-acid peptide isolated from bovine neutrophils, as a model of antimicrobial peptide-membrane interactions, and (2) NAP-22 peptide, a cationic myristoylated peptide crucial for neuronal growth and plasticity, as a model of protein-induced lipid recruitment. Using this combined AFM-fluorescence approach with supported planar bilayers, we showed that: (1) the attachment of a fluorescent dye molecule can alter the physical properties of the host lipid and inhibit the lipid-dye conjugate from associating with raft domains, (2) indolicidin preferentially associates with the more disordered fluid domain and induces gel domain lowering in a concentration- and lipid-dependent manner, and (3) NAP-22 peptide induces coalescence of raft domains at low peptide concentration and phase-immersion at high peptide concentration resulting the co-localization of cholesterol and phosphatidylinositol biphosphates. This work demonstrates the versatility of a combined AFM-fluorescence approach as well as its limitations of the current configurations.
