Studies of Protein-protein Interactions at Membrane Interfaces Using Correlated Atomic Force and Fluorescence Microscopy PDF Download
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Author: James Eric Shaw
Publisher:
ISBN: 9780494393802
Category :
Languages : en
Pages : 278
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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.
Author: James Eric Shaw
Publisher:
ISBN: 9780494393802
Category :
Languages : en
Pages : 278
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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.
Author: George Richard Heath
Publisher:
ISBN:
Category :
Languages : en
Pages : 504
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Book Description
Author: George Richard Heath
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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Book Description
Author: Peter Schuck
Publisher: Springer Science & Business Media
ISBN: 0387359664
Category : Science
Languages : en
Pages : 537
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Book Description
This volume successfully and clearly examines how biophysical approaches can be used to study complex systems of reversibly interacting proteins. It deals with the methodology behind the research and shows how to synergistically incorporate several methodologies for use. Each chapter treats and introduces the reader to different biological systems, includes a brief summary of the physical principles, and mentions practical requirements.
![Functional Force Mapping of Membrane-associated Protein Complexes [microform] PDF](https://books.google.com/books/content/images/frontcover/z-YMywAACAAJ?fife=w80-h100)
Author: Andrea-Lynn Slade
Publisher: National Library of Canada = Bibliothèque nationale du Canada
ISBN: 9780612918610
Category :
Languages : en
Pages : 602
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Book Description
Molecular recognition is governed by subtle structural and chemical cues between complementary binding partners. Understanding and ultimately controlling these interactions underpins efforts in fields from structural biology, biochemistry, to combinatorial chemistry and protein engineering. While traditional approaches to studying protein-protein interactions and molecular recognition phenomena have provided in-depth molecular scale details on equilibrium binding affinities and the local atomic structure of the binding interfaces, the actual force(s) involved in binding remain largely unknown. We have coupled the ability of scanning probe microscopy (SPM) to provide true three-dimensional images with its high force sensitivity into a new imaging modality termed 'topography targeted force mapping microscopy' (T2FMM) and have applied this technique to perform functional force mapping of membrane associated protein complexes. This approach allows us to physically map the local structure of the binding interface of a membrane protein or receptor and by using a ligand tethered to the scanning tip, measure the forces between the ligand and its complementary binding site as a function of spatial location. But do the observed binding forces necessarily reflect the forces associated with a fully functional membrane protein that is activated upon the binding of a ligand? To address this question, we have combined T2FMM with total internal reflectance fluorescence (TIRF) spectroscopy and microscopy to allow in situ correlation between (1) topography (AFM imaging), (2) interaction forces (AFM force measurements), and (3) functional activity (fluorescence) for individual membrane protein complexes. Our studies have demonstrated the ability of this technique to provide novel insights into the structure of both integral membrane proteins and membrane-associated proteins in a native membrane environment. It also allows the localization of membrane receptors within specific regions of a supported planar lipid bilayer (SPB) system, as well as the co-localization of receptor molecules and membrane-associated proteins upon binding to the membrane surface. Force mapping studies performed using T2FMM and the well-characterized model membrane-associated protein system of cholera toxin have now provided framework necessary to perform 'functional' force mapping of ligand-membrane protein complexes, such as the insulin-insulin receptor (IR) complex, by T2FMM, while simultaneously monitoring the activation of the IR upon insulin binding though correlated fluorescence measurements.
Author:
Publisher:
ISBN:
Category : Dissertations, Academic
Languages : en
Pages : 800
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Author: John Oreopoulos
Publisher:
ISBN: 9780494777190
Category :
Languages : en
Pages : 476
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Book Description
Biological membranes are heterogeneous two-dimensional fluids composed of lipids, sterols and proteins that act as complex gateways and define the cell boundary. The functions of these interfaces are diverse and specific to individual organisms, cell types, and tissues. Membranes must take up nutrients and small molecules, release waste products, bind ligands, transmit signals, convert energy, sense the environment, maintain cell adhesion, control cell migration, and much more while forming a tight barrier around the cell. The molecular mechanisms and structural details responsible for this diverse set of functions of biological membranes are still poorly understood, however. Developing new tools capable of probing and determining the local molecular organization, structure, and dynamics of membranes and their components is critical for furthering our knowledge about these important cellular processes that are often linked to health and diseases.Combinatorial microscopy takes advantage of the rich properties of light (intensity, wavelength, polarization, etc.) to create new forms of imaging that quantify the motions, orientations, and binding kinetics of the sample's biomolecular constituents. These new optical imaging modalities can also be further combined with other types of microscopy to produce spatially correlated micrographs that provide complementary pieces of information about the sample under investigation that would otherwise remain hidden from the observer if the two imaging techniques were applied independently. The first part of this thesis provides a detailed account of the construction of a specialized hybrid microscopy platform that combines polarized total internal reflection fluorescence microscopy (pTIRFM) with atomic force microscopy (AFM) for the purpose of studying fundamental sterol-lipid and antimicrobial peptide-lipid interactions in model membranes. The second half describes a combined pTIRFM and Forster resonance energy transfer (FRET) imaging method to elucidate the oligomeric state and spatial distribution of carcinoembryonic-antigen-related cell-adhesion molecules (CEACAMs) in the membranes of living cells.
Author: G. Ulrich Nienhaus
Publisher: Humana
ISBN: 9781617375255
Category : Science
Languages : en
Pages : 0
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Book Description
A readily reproducible collection of established and emerging techniques for studying the interaction between proteins and ligands, including biochemical/bulk techniques, structure analysis, spectroscopy, single-molecule studies, and theoretical/computational tools. Among the highlights are surface plasmon resonance (SPR) and reflectometric biosensor approaches, high-throughput screening with confocal optics microscopy, single molecule fluorescence and fluorescence correlation spectroscopy (FCS), atomic force microscopy (AFM), crystallography of reaction intermediates, and time-resolved x-ray crystallography. The protocols follow the successful Methods in Molecular BiologyTM series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
Author: Stefano Mangani
Publisher: Springer Science & Business Media
ISBN: 3642379990
Category : Science
Languages : en
Pages : 167
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Book Description
"Disruption of Protein-Protein Interfaces" reviews the latest developments and future perspectives in drug discovery at protein-protein interfaces. The authors detail experimental and computational tools to tackle the subject and highlight the contribution of the Italian research community to the field. Evidence shows that blocking or modulating protein-protein interactions might lead to the development of useful new drugs. Consequently, in recent years great effort has been dedicated to unveiling the molecular details of protein-protein interfaces by structural techniques e.g. X-ray diffraction, NMR spectroscopy. This book, written and edited by leaders in the field, provides examples from the literature of successes and failures to develop drug-like molecules effective in interacting at protein-protein interfaces.
Author: Nicholas Andrew Geisse-Sierralta
Publisher:
ISBN:
Category :
Languages : en
Pages :
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