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colleen colleen
wrote...
Valued Member
Posts: 17076
12 years ago
Trypsin is a hydrophilic protease and therefore is unable to cross the plasma membrane and enter a cell. However, it is capable of digesting the hydrophilic portions of membrane proteins. As a result of these properties, researchers investigating membrane proteins have treated cells with trypsin then separated the proteins by SDS-PAGE to gain insight into the orientation of particular proteins within the membrane. Describe an experiment using this information and the results you would expect with the following types of membrane-associated proteins. (Assume the proteins are single polypeptides.)

a. an integral membrane protein with the N-terminus exposed on the surface
b. an integral membrane protein with the C-terminus exposed on the surface
c. an integral membrane protein with three hydrophilic loops (only) exposed on the surface
d. a membrane-associated protein facing the cytoplasm
e. an imbedded membrane protein
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Sunshine ☀ ☼

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wrote...
Educator
12 years ago
The experiment would need to contain exposure of the cells to trypsin, followed by protein isolation and SDS-PAGE. Some students may wish to add amino acid sequencing in their answers as well. As for the expectations of results:

a. This protein would be shorter (therefore migrating further in the gel), and the N-terminal end would be missing.
b. This protein would be shorter (therefore migrating further in the gel), and the C-terminal end would be missing.
c. This protein would be cut into four smaller fragments (assuming the fragments are different in length), due to the digestion of the three extracellular loops.
d. This protein would not be exposed for digestion and would therefore be unaffected by trypsin. In the gel, this protein would remain the same size.
e. This protein would not be exposed for digestion and therefore would be unaffected by trypsin. In the gel, this protein would remain the same size.
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