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CBNS 101 lecture3 - Summary Lecture 2 1 Dark eld microscopy...

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2. Membrane structure and function: Lipid bi-layer Summary: Lecture 2 1. Dark field microscopy: Dyes, fluorescent proteins, fluorescence versus confocal microscopy, 3-dimensional reconstruction of cells and tissues (Membrane lipids and membrane proteins) 3. Membrane lipids: structure, types of lipids, fluidity of membranes, asymmetric distribution of lipids and its relevance, site of lipid synthesis 4. Membrane Proteins: Structure and function, distribution and mobility within membrane 5. Channels, carrier proteins Lecture 3
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Membrane proteins suspended directly in bilayer. “float” in “sea of lipid”. Fluid because proteins could move laterally in plane of membrane. Mosaic because membrane would have distinct regions Key issue was whether proteins (hydrophilic peptide backbone) could pass through the membrane’s hydrophobic zone. How are the protein molecules arranged in the membrane? 1972: S.J. Singer and G. L. Nicolson proposed fluid mosaic model
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Proteins span the lipid bilayer in an alpha-helical configuration Membrane spanned by regions with hydrophobic side chains Maximizes hydrogen bonding of polypeptide backbone-masks from lipid Polypeptide can pass through multiple times, resulting in defined orientation of protein in membrane.
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Integral membrane proteins-Released by reagents (detergents) that solubilize the bilayer Peripheral membrane proteins-Released by treatments that leave membrane intact. Lipid anchored membrane proteins-Covalent linkage to lipid molecule, can be released by phospholipases Properties of different membrane proteins
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Purification and reconstitution of membrane proteins
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Red -Fluorescence from antibody against mouse membrane protein Purple -Fluorescence from antibody against human membrane protein Induce cells to fuse with polyethylene glycol Do the two protein populations mix in the hybrid cell? Are proteins mobile in the membrane? Frye and Eddin J. Cell Sci., 1970
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Rate of membrane protein movement can be measured by FRAP method Rate of membrane protein movement can be measured by FRAP method -Measure the amount of time to recover fluorescence in area Fluorescent dyes attached to membrane lipids or proteins. Fluorescent dyes attached to membrane lipids or proteins. BLEACH WITH LASER BEAM
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Example of proteins restricted to particular domains Different monoclonal antibodies label cell surface molecules on different domains of a guinea pig sperm cell.
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Regulation of protein movement within membranes Apical membrane Lateral membrane Basal membrane Apical-basal movement is regulated by tight junction
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Cells can regulate lateral movement of proteins within a membrane domain A) Self assembly of proteins into aggregates (Bactereorhodopsin of Halobacterium) B) Tethering to macromolecules inside the cell (cytoskeletal attachment) C) Tethering to macromolecules outside the cell (agrin in nerve cells) D) Tethering to proteins on surface of another cell (adhesion proteins)
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Important points about membrane structure and function
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