Hydropathy analysis of a membrane protein's amino acid sequence predicts the protein's transmembrane segments and orientation in the membrane

Question

It is necessary to confirm those predictions with experimental analysis. One experimental approach for plasma membrane proteins is to use the protease trypsin to digest extracellular domains of these proteins. (Trypsin is hydrophilic and cannot cross the plasma membrane to enter the cell.) When added to cells, trypsin digests the hydrophilic portions of plasma membrane proteins exposed outside the cell into small fragments but will not have access to transmembrane or cytosolic regions of proteins. Design a general experiment using trypsin and SDS-PAGE to address membrane protein association and orientation, and describe specific expected results that would be consistent with the following predicted membrane protein associations and orientations:
a. a singlepass integral membrane protein with only its N-terminus exposed on the extracellular surface
b. a singlepass integral membrane protein with only its C-terminus exposed on the extracellular surface
c. a multipass integral membrane protein with only three hydrophilic loops exposed on the extracellular surface
d. a fatty acylated protein
e. a GPI-anchored protein

What will be an ideal response?

Answer 1

Answer: Experiment: Treat one set of intact cells with trypsin and use a second set of untreated cells as a control source of the full-length protein. Use SDS-PAGE to separate the total cell proteins or proteins in an isolated plasma membrane fraction from both the treated and untreated cells. Identify and compare the protein of interest and any of it proteolysis fragments in the treated and untreated control samples.
Expected results:
a. The undigested region of this protein (transmembrane segment and cytosolic C-terminus) would be shorter and therefore migrate further in the gel in the treated sample than in the control sample, because the trypsin would digest the extracellular N-terminal end.
b. The undigested region of this protein (transmembrane segment and cytosolic N-terminus) would be shorter and therefore migrate further in the gel in the treated sample than in the control sample, because the trypsin would digest the extracellular C-terminal end.
c. Trypsin would cut the three extracellular loops of this protein, leaving four smaller fragments (migrating further in the gel) in the treated sample.
d. The entire fatty acylated protein anchored to the outer monolayer of the plasma membrane is cytosolic and would be completely protected from trypsin proteolysis. This protein would be the same size in the treated and control samples.
e. The entire GPI-anchored protein anchored to the outer monolayer of the plasma membrane is exposed extracellularly and would be completely digested into rapidly migrating small fragments in the treated sample but would appear full length in the untreated sample.