Question

Hydropathy analysis of a membrane protein's amino acid sequence predicts the protein's

transmembrane segments and orientation in the membrane. 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

Answer 1

Auli- a) In this case, the membrane proluns amino and sequences passes throughout the cell membrane only once in all prodeins, the N-terminus is at the beginning of the protein's sequence, therefore for this case, you will have a part of it Exposed to the extracellular matrix, . The other part will be embedded in the cell membrane and the c- terminus will be inside the cell, in the plasma. The SOS-PAGE/ Western blot results will show two bands, the N-teemunus part that was exposed in the Extracellular space, and there fore cut by trypsin, and a c-termins part inside the cell. b) for this case, the SDS-PAGE / western blot results will show two bands, the c- terminus part that was exposed in the Extraullular space, and therefore cut by trypsin, and a c- terminus part inside the cell c) for this case, the SDS-PAGE (western blot will show between foue bands

d) In this case, the fatty acetylated proteins are proteins anchored by lipids to the cell membrane, which means the whole proteins asequence is in the extracellulat spall and the lipid linked to it is the one Embedded in the cell membrane acting as an anchor. Therefore a lots of bands will be shown as the whole protein will be exposed to be cut by trypsin. en The result for this case will be the same as the previous as this is a lipid- anchored protein, where the whole protein is exposed to the extracellular space. Please rate my Auswer.