Covalent fatty acid chain eg v src is attached to the

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Unformatted text preview: . v-Src is attached to the membrane via a myristoyl group linked to its N-terminal glycine. Proteins can also be palmitoylated allowing them to attach to the membrane. They can be purified by treating cells with detergents. Oligosaccharide linkage to phosphatidylinositol: e.g. Thy-1 is attached via a GPI (glycosylphosphatidylinositiol) linkage. Such proteins are usually only found on the extracellular side of the membrane. It can be purified by treating with a PI-specific phospholipase C. Peripheral protein: e.g. Phospholipase A2. It can be extracted by breaking up the electrostatic interactions using high salt concentrations or a high pH. Some peripheral membrane proteins need calcium ions to form electrostatic interactions 7.06 Spring 2004 PS 1 key 10 of 11 with the phospholipid head groups and so these proteins can be released by adding a calcium chelator. (b) Why is it that polypeptides that enter the bilayer tend to pass completely through it rather than changing direction midway? Changing direction midway would involve making a bend in the polypeptide backbone, which is not energetically favored as it decreases the number of hydrogen bonds formed. Therefore, from a thermodynamic viewpoint it is much better if a protein passes through the entire bilayer. (c) You have cloned a multipass transmembrane receptor and are trying to determine whether the N-terminus is on the cytoplasmic or the extracellular side of the bilayer. How would the following information help you determine this: (i) position of glycosylated residues (ii) a series of monoclonal antibodies whose epitopes have been mapped (iii) a recombinant form of the protein that has been tagged at the N-terminus (i) Glycosylated residues are always found on the extracellular portion of a transmembrane protein – in a future lecture on protein secretion and modification we will discover why this is. (ii) If you have a monoclonal antibody you could incubate cells that express the receptor with this antibody, wash away any unbound antibody, cross-link bound antibody and then add a secondary antibody coupled to a fluorescent molecule. Antibodies can’t diffuse thro...
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